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Cellutions
Vol 4: 2012The Newsletter orCell Biology Researchers
Take control.
A novel RNA detection reagent enables gene
expression analysis in living cells Page 9
CellASIC ONIX live cell imaging platorm or
neural stem cell microenvironment control Page 13
Assessing enriched murine CD4+ T cells
dierentiated towards eector T helper cell lineages
with the Scepter 2.0 handheld automated cell
counter Page 18
Product Review Autophagy: Mechanisms
and connections to apoptosis Page 26
To subscribe to the quarterly Cellutions newsletter,
please visitwww.millipore.com/cellquarterlynews
Microuidic perusion enables long-term cell
culture, precise microenvironment control and
gene expression analysis Page 3
EMD Millipore is a division o Merck KGaA, Darmstadt, Germany
http://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/reply/form/cellutions?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/reply/form/cellutions?cid=BIOS-C-EPDF-1007-1301-BS -
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PRODUCT HIGHLIGHT
Take control.Design dynamic experimentsthe CellASIC way.
Biology is so much more than DMEM/FBS, 37 C, 5% CO2.
Model experiments creatively and achieve true cultureconditions with the CellASIC ONIX Microuidic Platorm.Push the boundaries o your cell biology experiments in an
in vivo-like environment its easy to program automatedchanges to conditions and track cell responses with thisexible, intuitive platorm.
Read the articles on page 3 and page 13 o this issue.
The power is yours.www.millipore.com/CellASIC
http://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/life_sciences/flx4/cellasic_live_cell_imaging?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/life_sciences/flx4/cellasic_live_cell_imaging?cid=BIOS-C-EPDF-1007-1301-BS -
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Microuidic perusion enableslong-term cell culture, precisemicroenvironment control
and gene expression analysisPaul Hung, Cindy Chen, Philip Lee, Terry Gaige, and Alex Mok
EMD Millipore Corporation
IntroductionThe analysis o living cells in vitrois critical to
understanding basic biology, signaling pathways,
drug eects, and disease models. Current methods
provide excellent means to interrogate living cells
via biomolecular probes, uorescence microscopy,and genetic manipulation1. However, technology or
environment control o living cells during analysis has
not advanced signifcantly since the Petri dish. There is
a growing body o evidence to indicate that the cellular
environment, or niche, is just as important (or even
more critical) than genetic actors or determining cell
phenotype2. Thereore, a method or providing more
accurate, dynamic control o living cells has the potential
to dramatically advance the state-o-the-art or live cell
analysis3,4.
The CellASIC ONIX Microuidic Platorm, in conjunction
with the CellASIC ONIX Microuidic Plate, provides
perusion-based microenvironment control or long-
term, high quality, live cell microscopy (Figure 1). The
microuidic chamber recreates the physiologic mass
transport condition or optimized cell health. Four
upstream uidic channels allow controlled exposure o
the cells to dierent solutions during live imaging. The
plate can also be cultured in a standard incubator using
a dedicated gravity driven ow channel. The cells are
in contact with a #1.5 thickness (170 m) optical glass
surace, enabling high quality imaging using an inverted
microscope. An integrated microincubator system delivers
temperature and gas control to the microuidic chambers.
In this study, we used the M04S Microuidic Plate
to demonstrate long-term culture o adherent cells,
to create dynamic solution profles (media switching
and spatial gradient), to immunostain cells within the
microuidic chamber, and to analyze gene expression.
Because gene expression is inuenced by numerous
cell cultural parameters such as soluble biochemical
actors, extracellular matrices, and stability o the
microenvironment, accuracy and physiological relevance
o gene expression analysis can be enhanced by
perorming such analyses in a dynamically controlled,
bio-inspired, microuidic system. Thereore, we
demonstrate here the ability to conduct gene expression
analysis using the CellASIC ONIX Microuidic Platorm
with M04S Microuidic Plates. Ultimately, directly
correlating gene expression patterns to phenotypes
observed during live cell imaging can provide powerul,
meaningul unctional genomics data, revealing
signaling networks and novel biomolecular interactions.Using breast cancer cell line MCF10A, we quantifed
the expression o Epidermal Growth Factor Receptor
(EGFR) using standard quantitative reverse transcription
polymerase chain reaction (qRT-PCR) techniques.
A BFigure 1.
Cells cultured in theM04S microuidicchamber. (A) HT-1080
cells imaged underphase contrast and (B)immunostained or nuclei(blue), actin (red), andmicrotubules (green).Fixing and staining wereperormed within themicrouidic chamber.Images were acquiredwith a 40X objective lens.
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Materials and MethodsThe CellASIC ONIX Microuidic Platorm
The M04S Microuidic Plate has a 96-well ootprint to
ft to typical microscope stage holders. The microuidic
chamber (our in each plate) has been designed to be
compatible with standard culture methods, including
surace coating, 3D culture, and co-culture. The bottom
surace o the chamber is plasma-activated glass, which
supports most standard cell lines without coating,
including HeLa, NIH3T3, MCF7, MCF-10A, HT1080,
HUVEC, and others.
The custom well layout maximizes live cell imaging
capabilities. The M04S has our independent ow units
(A-D), with each unit containing eight inlet and outlet
wells (one gravity inlet, our switching inlets, one cell
inlet, two waste outlets). The our cell culture chambers
are centralized under a single large imaging window
(Figure 2). The chamber-to-chamber distance is
5.2 mm, reducing objective travel time and ocus drit.The bottom surace o the plate is a #1.5 thickness
(170 m) optical glass slide to maximize quality o high
resolution, high numerical aperture imaging. The plate
houses all experiment solutions, allowing control with
an external pneumatic maniold (Figure 3). The maniold
lets the user direct ow rates and select exposure
solutions without perturbing the microscope stage. A
gas line allows control o the environment within the
microchambers through a network o gas-permeable air
diusion channels. Temperature is regulated through an
on-board heater/chiller on the maniold.
Figure 3.
Side view schematic o the microuidic plate with micro-incubation maniold on a microscope stage. The bottomsurace o the microuidic plate is a thin glass sheet, allowinghigh quality cell imaging. The plate is sealed to a pneumaticmaniold, allowing user control o the ow profle duringimaging. Additional air channels allow control o the gasenvironment.
Figure 2.
The M04S plate contains our independent ow units (A-D),each with our upstream solution inlets, a gravity ow inlet,a cell inlet, and two waste wells. The culture chamber is2.8 mm in diameter (120 m height) and is surrounded witha microabricated perusion barrier. Inlet 1 is a gravity owwell, allowing long term cell culture in a standard incubatorwithout a pressure system. Continuous ow o solutions rominlets 2-5 creates a dynamic exposure profle during live cellimaging.
A
D
C
B
1 765432 8
5.2 mm
Imaging Window
Flow Inlets (2,3)
Cell Inlet (6)Cell Culture Chamber
(2.8 mm diameter)Waste(7-8)
PerfusionBarriers
Unit Marker
Air Channels
Gravity Inlet (1)
Flow Inlets (4,5)
Gravity Inlet (1)
Gas-permeablemembrane
Cells
Recirculatinggas mixture
Manifold
Air pressure-driven flow
Convective Peltierheat exchanger
#1.5 thickness(170 m) glass
Glass window
MicrofluidicPlate
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Cell loading
Cells are loaded into the culture chamber using a capillary-
driven method. This allows the user to load cells using a
pipette, and can be done in a sterile laminar ow hood
without any external systems. The cell suspension is
pipetted into the cell inlet well (6) and liquid is aspirated
out o the waste wells (7 and 8). This creates a surace
tension orce that pulls the cell suspension into the
chamber. As the cells enter the chamber, the ow profle
allows them to settle to the oor without any stress.
Typically, loading into the microchambers occurs within
3 minutes and cells are allowed to settle or up to 30
minutes. I more cells are desired, higher concentrations
(or repeated loading cycles) can be implemented (Figure 4).
Figure 4.
Cell loading in the microuidic chamber. (A) Each chamber canbe seeded with up to a ew thousand cells by varying the celldensity during capillary ow loading. Images taken with a 4Xobjective o HT-1080 cells loaded at (B) 0.5, (C) 1.0, (D) 2.0,and (E) 4.0 million cells/mL.
Ater the cells settle to the bottom o the chamber,
continuous ow o medium enables them to attach and
grow. The chamber surace can be coated prior to cell
loading by owing the coating solution (e.g., poly-D-
lysine, collagen, fbronectin) into the chamber using
the same method. Alternatively, three-dimensional (3D)
culture can be achieved by mixing the cell suspension
with a gel matrix and loading together, allowing thecells/gel to polymerize within the chamber. The design
o the ow channels allows continuous perusion even
in the presence o 3D gel.
Gene expression analysis
Non-neoplastic human mammary MCF-10A epithelial
cells (obtained rom ATCC (CRL-10317) ) were grown in
Dulbeccos Modifed Eagle Medium F-12 (DMEM/F-12)
supplemented with 2% horse serum (Gibco), 5 ng/mL EGF
(Invitrogen), 0.5 g/mL hydrocortisone (Sigma), 100 ng/mL
cholera toxin (Sigma) and 10 g/mL insulin (Gibco) ina 5% CO
2atmosphere at 37 C. Prior to cell loading,
MCF-10A cells were prepared in a cell suspension o 1 X
106 cells/mL in the growth medium. Upon opening the
M04S microuidic plate packaging, priming solutions
were aspirated rom medium inlet well 1, cell inlet
well 6, medium outlet well 7 and cell outlet well 8.
Ater 10 L o cell suspension was loaded into well
6, medium in well 7 was aspirated again to distribute
cells evenly by capillary action. Plates were then laid
at in the incubator or an hour which allowed cells
to settle without any ow disturbance. Ater one hour
o incubation, 300 L o growth medium was added to
medium inlet well 1 and 50 L o the culture medium
into well 7 to initiate gravity driven perusion o growth
media during cell culture. Plates were then placed in the
incubator until cells were conuent.
To lyse the cells, medium was aspirated rom medium
inlet well 1, medium outlet well 7 and 8, ollowed by
pipetting 50 L o 1X PBS (Cellgro) into well 1. Ater
sealing the plate to the maniold, the CellASIC ONIX
FG Sotware was used to apply 4 psi on well 1 or 1
minute to rinse the channels and wash out cell debris inthe chambers. The maniold was then unsealed and all
liquid in well 1, 7, and 8 was aspirated. Then, 50 L o the
RNA extraction RNeasy RLT buer (Qiagen) was added
to well 1. Ater sealing the plate to the maniold, a cell
lysing program was initiated on the CellASIC ONIX FG
Sotware (4 psi on well 1 and 0.25 psi on well 6 or 20
seconds). We then unsealed the maniold and collected
20 L o the cell lysates rom well 7. RNA was extracted
using the RNeasy mini kit (Qiagen), ollowing the RNA
extraction procedures instructed in the manuacturers
manual.
B
A
D
C
E
4000
3000
No.
Cellsloaded
Cell Suspension (106 cells/mL)
2000
1000
29
0 1 2 3 4
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Total RNA was then synthesized into cDNA using
iScript cDNA synthesis kit (Bio-Rad) with random
hexamer primers. Quantitative PCR was perormed
with the ollowing primers: EGFR orward
(5- GGCAGGAGTCATGGGAGAA-3) and reverse
(5-GCGATGGACGGGATCTTAG-3). Samples were
normalized using oligonucleotides specifc to
RNA encoding the house-keeping gene, GAPDH:
orward (5-ACCCACTCCTCCACCTTTG-3) and reverse(5-CTCTTGTGCTCTTGCTGGG-3). cDNA was diluted 1:25
or GAPDH and 1:2 or EGFR beore qPCR. Quantitative
PCR was perormed using SsoFast Evagreen Supermix
(Bio-Rad) on the MiniOpticon Real-Time PCR Detection
System (Bio-Rad). The conditions or PCR were 95C,
3 minutes ollowed by 40 cycles o 95C 5 seconds, 60C
10 seconds. Analysis variance and post hoc tests o
quantitative PCR results were carried out using Bio-Rad
MiniOpticon sotware.
ResultsSolution switching
A key eature o the M04S plate design is that solutions
can be changed during live cell imaging without
perturbing the plate or microscope. This enables tracking
o cell responses to changing solution environments.
The M04S allows our dierent solutions to be switched
during the course o the experiment.
Exposure solutions are introduced rom the our inlet wells
and ow through the chambers to the waste (well 7). Wells
7 and 8 are connected as a shared outlet or increased
volume. Well 1 is a gravity ow well, with a perusion
rate o approximately 80 L/day. This is used or pre-
culture o cells in the M04S plate in a standard incubator
when solution exchange or imaging is not necessary; or
example, to expand or dierentiate cells over a period o
a ew days. Wells 2-5 are the pressure-driven wells. Theow rate and exchange times are given in Figure 5. The
highly laminar ow profle means that when the input
solution is changed, a sharp uid interace is created
that moves across the culture area rom let to right. The
velocity o this ront is given in Figure 5A. The actual local
exchange time (the transition rom solution 1 to 2 around
the cells) happens quickly, typically in a ew seconds. The
small volume o the culture chamber enables ast solution
exchange at ow rates rom 5-80 L/hour. This means that
a typical imaging experiment (with 300 L per inlet well)
can run or up to 72 hours.
As one example o ow switching, two solutions
(phosphate-buered saline (PBS) and dextran-conjugated
uorescein, 3 kDa, Invitrogen) were switched at 10-minute
intervals (Figure 5B). Note the rapid and complete response
o the solution, creating a clean step unction in the
culture region. Since all our channels converge near the
culture chamber, the M04S plate minimizes the dead
volume during switching to a ew nanoliters.
In addition to ow switching, the CellASIC ONIX
Microuidic Platorm was used to create spatial
gradients across a chamber (Figure 5C). When more
than one channel was owed simultaneously, laminar
ow and diusion across the interace created a stable
spatial gradient. For sensitive kinetic experiments, it is
recommended that a tracer dye be used to accurately
ollow solution ow profles.
Figure 5.
A) Flow rate through the chamber as a unction o pressure applied by the CellASIC ONIX Microuidic Platorm to the inlet wells (V2 V5) o M04S plate. Theow rates allow long-term, continuous ow experiments to be perormed on the microscope stage. The inlet wells hold 350 L o solution, allowing a singleexperiment to run or up to 72 hours. In general, a ow pressure o 1 psi is suitable or nutrient exchange. (B) Sequential switching between uorescent dye andPBS. Intensity was measured in the center o the culture region every minute with ow at 2 psi rom 2 channels. Data was plotted normalized to the max/min ointensity images. (C) Spatial gradient in the M04S chamber o uorescein conjugated dextran (3kDa). Intensity was measured in the y-direction o the chamber.
Light green line shows ow rom 2 channels simultaneously o 0% and 100% dye at 0.5 psi each. Dark green line shows ow rom 4 channels simultaneouslyat 0%, 25%, 50%, and 100% dye at 0.5 psi each. Di usion across the interaces creates a smoothed profle. Flow at aster rates will lead to sharper, step-likeboundaries.
A
Pressure (psi)
FlowR
ate(L/hr)
Wells 2-5
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150160
170
180
0 1 2 3 4 5 6 7 8 9
1.0
0.8
0.6
0.4
0.2
00 20 40 60
Minutes
B
Intensity
1.0
0.8
0.6
0.4
0.2
00 400 800 1200 1600
C
Microns (y-direction)
NormalizedIntensity
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Long-term cell culture
MDA-MB-231 cells were grown using the CellASIC
ONIX Microuidic Platorm in M04S perusion plates or
three days with continuous perusion. Figure 6, showing
the cells at our dierent time points, illustrates that cell
health and morphology were preserved or the entire
length o the experiment.
Time lapse imagingThe avorable cell culture environment in the M04S
chamber allows long term maintenance o adherent cells
under well-controlled conditions. This enables enhanced
live cell tracking o cellular events in response to changes
in media, cell cycle, drug dosing, and other stimuli. We
demonstrated two common methods or high resolution
cell analysis using microscopy: 1) immunostaining, and
2) transection. To acilitate immunostaining o cells
cultured in the M04S plate, an automated ow protocol
was set up to sequence fxing, permeabilizing, blocking,
primary antibody, secondary antibody, and all washsteps. This enabled monitoring o live cells in the M04S
plate, and then subsequently fxing the same cells and
analyzing by immunouorescence.
In a second example, live cells cultured in the M04S plate
were transected with GFP-tubulin (Invitrogen Cellular
Lights reagent). Ater exposure to the GFP reagent, the
cells expressed the protein o interest and could continue
to be tracked in the microuidic system (Figure 7).
Figure 7.On-chip staining o cells cultured in the M04S chamber. (A) HeLa cells cultured in themicrouidic chamber immunostained via ow exposure. Actin is shown in red, tubulin is shownin green. (B) MCF-10A cell cultured in the microuidic chamber and transected with GFP-tubulin via ow exposure. Images taken with a 100X objective lens.
A B
Figure 6.
Growth o MDA-MB-231 cells in the microuidic chamber withcontinuous perusion ater (A) 1 hour, (B) 1 day, (C) 2 days, and(D) 3 days. Images were acquired with a 4X objective lens.
A
C
B
D
Figure 8.
Images o the cell culture chamber beore (let) and ater (right) cell lysis. The cells in the imageat let were allowed to grow to conuency.
Gene expression analysisWe frst quantifed the total number o MCF-10A cells
cultured to semi-conuency in each chamber o the
our-chamber M04S plate using microscopy and Image
J sotware (http://rsbweb.nih.gov/ij/). Ater counting the
cells, we used an in-well lysis procedure and then removed
the lysates or analysis. To demonstrate the efciency
o the lysis protocol, we grew cells to near conuency in
the M04S Microuidic Plate and imaged the cell culture
chamber beore and ater lysis (Figure 8). The clear
chamber ater lysis indicated high collection efciency,
with no remaining cells let over in the chamber.
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Figure 9.
(Top) Per-cell EGFR expression normalized to GAPDHexpression and total number o cells per chamber. (Bottom)Comparison o per-cell EGFR expression o MCF-10A cellscultured in the CellASIC ONIX Platorm (M04S plate) vs. instandard Petri dish.
Per Cell EGFR Expression(Microfluidics)
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
A B C D
Chambers
Norma
lizedExpressionValues
Per Cell EGFR Expression(Microfluidics vs. Petri Dish)
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
Dish Microfluidics
NormalizedE
xpressionValues
SummaryThe ability to control and monitor living cells is critical or understanding signaling
networks and complex phenotypes in response to stimuli. We have developed the
innovative CellASIC ONIX Microuidic Platorm using the M04S perusion chamber
plate to optimize cell microenvironment control while acilitating long-term, high
quality microscopy. This design has been demonstrated with a wide range o cell lines
or uorescence quantifcation, solution exchange response, and time-varying inputs.
Existing cell analysis methods such as immunostaining, transection, uorescent
probes, and more, are easily adapted to the microuidic ormat. Further, the ease o
use, exibility, and accessibility o this advanced technology platorm should prove
benefcial to a wide range o cell biology applications, including accurate, meaningulgene expression analysis.
Reerences1. Wessels JT, et al. Advances in cellular, subcellular, and nanoscale imaging in vitroand in vivo.
Cytometry A. 2010 Jul;77(7):667-76.
2. Bissell MJ, Radisky D. Putting tumours in context.
Nat Rev Cancer. 2001 October;1(1):46-54.
3. Lee P, Gaige T, Hung P. Microluidic systems or live cell imaging. Methods Cell Biol.
2011;102:77-103.
4. Lee PJ, Gaige TA, Hung PJ. Dynamic cell culture: a microluidic unction generator or live cell
microscopy. Lab Chip 2009 Jan 7;9(1):164-6.
Description Catalogue No.
CellASIC ONIX Microuidic System Package includes CellASIC ONIX Microuidic System, Maniold,
Accessory Box, and CellASIC ONIX FG Sotware EV262
CellASIC ONIX Microincubator Package or Temperature and Gas Control: Includes CellASIC ONIX
Microincubator Controller, Microincubator Maniold, and Accessory BoxMIC230
M04S Microuidic Switching Plate or Mammalian Cells (4 Chambers) M04S-03-5PK
Available romwww.millipore.com.
FEATURED PRODUCTS
By conducting RT-PCR analysis o the collected lysates, we quantifed EGFR and GAPDH
gene expression in the cells. The EGFR expression per cell was normalized to GAPDH and
total cell number in each chamber (Figure 9, top). The same experiment was perormed
on cells cultured on standard petri dishes. We ound an almost 3X dierence in per cell
EGFR expression between the M04S plate and a standard petri dish (Figure 9, bottom).
While we do not know the cause o this increase, it was highly reproducible and may
reect the environmental conditions supported in the microuidic chambers.
http://www.millipore.com/catalogue/item/EV262?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/MIC230?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/M04S-03-5PK?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/M04S-03-5PK?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/MIC230?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/EV262?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/M04S-03-5PK?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/MIC230?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/EV262?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BS -
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A novel RNA detection reagentenables gene expression analysisin living cellsDon Weldon, Erika Wells, James Graham, Laura White, Kevin Su, Alex Ko,
Yuko Williams, Martin Santillan, Jessica OConnor and Matthew Hsu
EMD Millipore Corporation
IntroductionDetecting gene expression has traditionally been limited
to technologies that examine expression in lysed or fxed
cell populations. The ability to detect RNAs in individual,
live cells can enable an unequivocal assessment o gene
expression changes that occur in direct response tospecifed perturbations. Determining which genes are
up- or down-regulated in these perturbed cells provides
insight into the relationships between gene expression
networks and cell unctions.
We present the SmartFlare detection reagent
(Figure 1), a novel probe capable o detecting specifc
mRNAs and miRNAs in live, intact cells1. This technology
allows or carrier-ree cellular endocytosis o the
reagent, ollowed by detection and relative quantitative
analysis o RNA levels. Because the reagent leaves thecell ater the detection event, the same sample can
be used or any downstream analysis, enabling the
measurement o multiple biomarkers or downstream
unctionalities in the same cells. Additionally, this
reagent requires no upront sample preparation, has no
toxic eects on cellular ate and no known nonspecifc,
o-target eects. Compared to currently used methods
or interrogating RNA that involve examination o
non-native, amplifed RNA targets, SmartFlare probes
have the potential to provide results that show greater
correlation to in vivoobservations. In vivo, most cells
reside in heterogeneous tissues, and cell-to-cell variation
in gene expression can be subtle yet crucial or tissueunction. SmartFlare technology can be used to
quantitate RNAs in individual cells, providing heretoore
unobtainable gene expression inormation that
distinguishes one heterogeneous cell population rom
another with high resolution.
Figure 1.
SmartFlare RNA Detection Probe
Gold-quencheddye on reporterstrand
Goldnanoparticle
Capture strand/reporter strandduplex
-
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Materials and Methods
Figure 2 shows the structure o a SmartFlare probe and
its mechanism o action. Each probe consists o a gold
nanoparticle conjugated to many copies o the same
double stranded oligonucleotide encoding the target
sequence. One strand o the oligonucleotide bears a
uorophore that is quenched by its proximity to the gold
core. When the nanoparticle comes into contact withits target, the target RNA displaces the uorophore. The
reporter strand, now unquenched, uoresces and can be
detected using any uorescence detection platorm.
Wavelength (nm)
Intensity
(C
PS)
Cy5 fluorescence (probe + target-specific oligo)
Cy5 fluorescence (probe + nontarget oligo)
Cy5 fluorescence (probe alone)
0
50000
150000
650 700 750
100000
Figure 3.
Specifc detection otarget RNA sequences.
The SmartFlare probealone (in buer) generateda background level ouorescence (pink).Following addition oa noncomplementaryoligonucleotide, theuorescence signal (red)was unchanged. Uponaddition o a complementarysequence, the uorescencesignal (yellow) reached itsmaximum intensity.
Assay Protocol
The ollowing general protocol is used or SmartFlare
RNA Detection Probes; however, concentrations o
probe, incubation times and detection methods can vary
depending on the specifc cell types and probes used.
1. Culture cells to 60-80% conuency
2. Add SmartFlare probe3. Incubate overnight (16 hours)
4. Detect via ow cytometry, microscopy,plate reader, or any other uorescent
detection platorm
Results
As shown in Figure 3, a typical SmartFlare RNA
Detection Probe exhibits specifcity or its target, as
evidenced by the increase in uorescence upon addition
o the target sequence, and lack o signal when a non-
target sequence is added in equal amounts.
Figure 2.
Molecular mechanismo SmartFlare RNADetection Probe
The oligo duplexes are designed withan RNA capture sequence and a
complementary reporter sequence.
In absence o target, probeis quenched by gold and does
not uoresce
When Target is present it will bindthe capture strand, releasing
the reporter strand, which then uoresces.
Target
+
.
Capture Strand
Reporter Strand
.
-AAAAAAATCAACCATACACCGTGACTTTGCTTGACCC
-AGTTGGTATGTGGCACT
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0
100 101 102 103 104
10
5
25
20
15
40
35
30
Count
miR-21 Cy3 (YEL-HLog)
HEK-293 DU-145
DU-145 HEK-293
Fluorescence(M5-510)
Cycles
1 2 6 9 10 1613 19 22 25 29 4034 37314
Fluorescence(M5-510)
Cycles
30
40
50
60
70
80
90
100
110
40
50
60
70
80
90
100
110
1 2 6 9 10 1613 19 22 25 29 4034 37314
MCF-7No probe HeLa
M CF-7 HeLa
0
20
100 101 102 103 104
40
60
80
100
120
140
EGFR mRNA probe (RED2-HLog)
FGF2 mRNA probe (RED2-HLog)
Count
No probe HT1376
0
100 101 102 103 104
40
20
60
80
100
120
140
160
Count
HUVEC
HT1376 HUVEC
180
Sample application: miRNA quantitationto distinguish two cell types
Because miRNAs are potent regulators o gene
expression, requently determining cell ate, they can be
eective biomarkers or disease states. Cell types can
requently be distinguished comparing relative levels o
specifc miRNAs. A probe to miR-21 was developed to
test the ability o SmartFlare technology, in conjunctionwith ow cytometry, to detect dierences in expression
between two cell types: HEK-293 cells (expressing low
levels o miR-21) and DU-145 cells (expressing high
levels o mIR-21). Low expression was indeed seen in the
HEK-293 cells (Figure 4, ar let section o the histogram)
and DU-145 cells showed a distinctly higher expression
(Figure 4, ar right o the histogram).
SmartFlare quantitation correlates withresults rom quantitative PCR (qPCR)
By treating cells with SmartFlare probes and measuring
subsequent uorescence with ow cytometry, we
measured the levels o two mRNA targets, EGFR and
FGF2, in cell types that show dierences in expression
o each o these targets (Figure 5, let). To confrm
expression levels, we quantitated the same RNAs in these
cells using reverse transcription PCR (RT-PCR). Because
SmartFlare technology allows or downstream assays
ollowing detection and analysis, RT-PCR was perormed
on the same cells to compare the relative amounts o
target RNA in each cell type (Figure 5, right).
Figure 4.
Target-specifc miRNAdetection using owcytometry. Detection o amicro RNA target miR-21 inboth HEK-293 cells (typicallyused as a low expresser omiR-21) and in DU-145 (aprostate cancer cell lineknown to express high levelso miR-212) using owcytometry. The histogramshowing the HEK-293 cellpopulation is shown overlaidwith the histogram o theDU-145 cell population,reecting two distinctpopulations o cells based ontheir gene expression profle.
Figure 5.
Probe detection o mRNAlevels correlates to RT-PCRdata. Using SmartFlaretechnology to determine themRNA levels o EGFR mRNA(A) in HeLa and MCF-7 cellsas well as FGF2 mRNA (B)in HUVEC and HT1376 cells,both o which correlate totheir RT-PCR levels. Flowcytometry provides addedinormation at the singlecell level as well as how theexpression is distributedwithin the population.
As expected, the cells that yielded lower Ct values
correlated with higher gene expression by ow
cytometry, indicating that the higher the level o RNA
target present is reected in the histogram as having a
higher mean uorescence intensity.
The histograms revealed additional inormation on the
variation o the expression within each cell population.
For example, the HeLa cells (sharp light green peak)
showed much more uniorm expression o EGFR than
MCF-7 cells (wider dark green peak).
A.
B.
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Fluorescence microscopy ollowingSmartFlare treatment reveals geneexpression dierences
Fluorescence microscopy was perormed on HeLa and
MCF-7 cells ollowing addition o either an EGFR probe or
a scrambled sequence (Figure 6). Both cell types showed
low levels o uorescence with scrambled sequence. The
HeLa cells showed a dramatic increase in uorescence with
the EGFR probe compared to the MCF-7 cells. The results
correlated with our ow cytometry and RT-PCR data.
be reused ollowing analysis and ater cell sorting or
additional downstream experiments on those same cells.
By enabling the researcher to discover cells that express
particular genes at particular levels in real time, thistechnology can enhance the signifcance o observed links
between genotype and phenotype in heterogeneous cell
populations, truly enhancing the value o data obtained
or RNA analysis.
Reerences1. Prigodich AE et al. Nano-Flares or mRNA Regulation and Detection.
ACS Nano. 2009 August 25; 3(8): 21472152.
2. Jinsong Li, MiR-21 Indicates Poor Prognosis in Tongue Squamous Cell
Carcinomas as an Apoptosis Inhibitor, Clin Cancer Res 2009; 15:3998
3. Prigodich AE et al. Multiplexed nanoares: mRNA detection in live cells.Anal Chem. 2012 Feb 21;84(4):2062-6.
Conclusions
We have shown that this novel live cell RNA detection
technology is easy to use and robust. These results
demonstrate that this reagent is sensitive and specifc
the SmartFlare probe emits uorescence only uponaddition o its complementary target sequence. It can
detect both micro and messenger RNA in the cytosol o
living cells and has shown good correlation to qRT-PCR.
Furthermore, the probes are platorm-agnostic, enabling
quantitation via ow cytometry or via microscopy,
without amplifcation.
The many potential uses o this breakthrough
technology include sorting o cells based on gene
expression (enabling even higher levels o enrichment
using additional intracellular markers), live celltracking o RNAs, and detection o multiple types o
biomolecules (such as protein and RNA) in the same
sample. Previous reports have already confrmed that this
technology can be used or multiplexed detection o up
to three dierent RNAs (using dierent uorophores),
enabling the normalization o gene expression levels
to housekeeping or control genes within individual
cells3. Notably, SmartFlare technology allows cells to
Scrambled Control
MCF-7Cells
HeLaCells
EGFR Target
Figure 6.
EGFR probe detection inhigh and low expressingcell lines. EGFR expressionin MCF7 and HeLa cellscompared to a scrambledsequence control. Specifcityor the target o interest is
evident in the uorescenceintensity o the EGFR probesin the HeLa cells vs theMCF-7 cells.
Description Catalogue No.
EGFR SmartFlare Probe SF-116
FGF2 SmartFlare Probe Coming soon
Scrambled Control Cy5 SmartFlare Probe SF-102
miR-21 SmartFlare Probe Coming soon
guava easyCyte 8HT Flow Cytometer 0500-4008
Available romwww.millipore.com.
FEATURED PRODUCTS
http://www.millipore.com/catalogue/item/SF-116?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SF-102?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/0500-4008?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/0500-4008?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SF-102?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SF-116?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BS -
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AbstractNeural stem cells (NSCs) are sensitive to microenvi-
ronmental cues, including cell-cell contact, cell-ECM
interaction, nutrient and waste transport, as well as
environmental oxygen composition. However, how
these parameters in the microenvironment aect the
stem cells morphology, prolieration, and dierentia-
tion remains an open area or research. In this study,
we demonstrated how the CellASIC ONIX Microuidic
Platorm with its microuidic cell culture devices are
capable o multi-parametric microenvironment control
or NSC studies.
IntroductionNSCs are sel-renewable and multipotent cells that are
capable o generating various phenotypes o the nervous
system1
. First described in the subventricular zone othe adult mouse brain2, NSCs have received considerable
attention due to their potential or therapeutic use.
Research eorts generally divide into three ocus areas:
expansion, dierentiation and cell:cell interactions.
While commercial sources or NSCs have grown rapidly,
building a physiologically relevant in vitroNSC culture
model to thoroughly study the biology behind NSC
expansion, dierentiation and cell:cell interactions
remains challenging. In addition to two-dimensional
(2D) culture asks, researchers have attempted other
culture technologies such as three-dimensional (3D)
neurospheres3, scaolds4, and microuidics5. While each
technology has separately shown that NSCs are sensitive
to microenvironmental cues, there has been no unifed
platorm enabling researchers to systematically control
the microenvironment or NSC culture.
Figure 1.
(Above) CellASIC ONIX Microuidic Platorm with themicrouidic system, the microincubator controller andmicroincubator maniold. (Right) Layout o M04S plate(our independent units and eight wells per unit).
CellASIC ONIX live cell imagingplatorm or neural stem cellmicroenvironment controlPaul Hung, Cindy Chen, Philip Lee, Terry Gaige, Brandon Miller,
Andrew Zayac, Ivana Zubonja and Alex Mok
EMD Millipore Corporation
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The CellASIC ONIX Microuidic Platorm oers
comprehensive cellular microenvironmental control or
cell culture studies (Figure 1). The ability to control
multiple parameters has been integrated into the
CellASIC ONIX Microuidic Platorm to enhance the
cellular microenvironment or NSC culture. To modulate
the degree o cell-cell contact, the system can be set up
to seed cells at varying density, leading to varying spatial
distribution. To enable changes to cell-ECM interaction,we developed protocols to coat the #1.5 coverglass
substrate with polyornithine/laminin, poly-D-lysine or
poly-L-lysine. For nutrient and waste transport, both
passive gravity-driven and pneumatically-driven control
can provide low-shear, steady-state perusion through
NSC cultures with solution switching. Since the plate
containing the cell culture chambers is made o
gas-permeable materials, it is possible to inoculate NSC
cell culture with dierent mixtures o gases. In this study,
we show that the oxygen microenvironment can be
tuned to range rom severe anoxia to hyperoxia. Tocharacterize NSCs grown in the microuidic-controlled
microenvironment, an automated immunostaining
protocol was developed to visualize nestin and Sox2
within NSC cell culture.
We also tested the speed at which gas conditions could
be changed using the microincubator, to assess the utility
o the system or studying cell responses to hypoxia.
The successul combination o environment control with
perusion culture in a microuidic platorm promises to
urther close the gap between in vitroexperiments and
in vivorelevance.
Materials and MethodsMaterials. Adult rat hippocampus neural stem cells and
antibodies recognizing Nestin and Sox2 were obtained
rom EMD Millipore (Cat. Nos. SCR021 and SCR022).
The CellASIC ONIX Microuidic System (Cat. No. EV262),
Microincubator Controller (Cat. No. MIC230), Tri-Gas
Mixer (Cat. No. GM230), and Microuidic Plates
(Cat. No. M04S-03-5PK) were also purchased rom
EMD Millipore. Poly-L-ornithine and laminin wereacquired rom Sigma. For poly-L-ornithine, the stock
solution was prepared with sterile, deionized water to a
fnal concentration o 50 g/mL. Laminin was prepared
with 1x phosphate-buered saline (PBS) at 7 g/mL.
The coating solutions were stored at 4 C. All other
reagents, such as rat neural stem cell culture medium
(Cat. No. SCM009) and basic fbroblast growth actor
(Cat. No. GF003) were obtained rom EMD Millipore. For
uorescent microscopy, an Olympus IX-71 inverted
uorescence microscope with an automated stage was
used. To validate the oxygen concentration in the cell
culture chamber, an oxygen sensor rom Presense was
attached inside the microuidic device.
Coating the cell culture chamber with extracellular
matrices. Liquid was aspirated rom wells 1, 6, 7, and 8
without aspirating liquid rom the inner rings (see user
guide or detailed protocol)6. 300 L o poly-L-ornithinesolution was then added to well 6. The plate was then
placed inside a traditional cell culture incubator at 37 C
or 24 hours to coat the M04S microchamber. Ater 24
hours, the plates were retrieved and liquid in wells 6, 7
and 8 was aspirated. 300 L o sterile, deionized water
was used to rinse the well beore another 300 L o
sterile water was added to well 6 to wash the
microchamber. The plate was then placed in an incubator
or 4 hours. For the second layer coating o laminin, the
water in wells 6 and 7 was aspirated. 300 L o the
laminin solution was then added to well 6. The plate wasreturned to the incubator or 24 hours. Beore seeding
the cells, the laminin solution in wells 6 and 7 was
aspirated. 300 L o 1x PBS was added to well 1 and
well 6. The device was then returned to the incubator or
2 hours to allow the PBS to gently wash out the
remaining laminin solution in the microuidic plate.
Cell seeding. The cell suspension was prepared
according to the vendors protocol and seeded at
both high and low densities. For low density seeding,
1 x 105/mL was recommended. We seeded cells at high
density (1 x 106/mL) to allow close cell-cell contact.
Beore introducing the cells into the microuidic device,
the liquid in well 1, 6 and 7 were frst aspirated. The inner
rings in well 6 and 7 were then careully aspirated
(extended aspiration will result in bubble introduction
into the microuidic channel). 10L o the cell
suspension (1,000 total cells or low cell density and
10,000 total cells or high cell density) was then added
into the inner ring o well 6. The plate was then placed in
a laminar cell culture hood or 30 minutes to allow the
cells to load into the microchamber and settle.
Gravity-driven perusion culture. To stabilize the cells,
300 L o the culture medium was added to well 1 to
allow gravity-driven perusion and the plate was placed
inside a regular incubator. For prolonged culture in the
incubator, the medium in wells 1 and 7 was aspirated
every 48 hours, and 300 L o the culture medium was
added into well 1 to re-establish gravity-driven perusion.
To culture the cells on the microscope, the PBS was
aspirated rom wells 2, 3, 4 and 5 but not the inner rings.
As a guideline, 300 L in each well, when owed at 0.5
psi, could provide ample nutrients to the cell cultures or
up to 36 hours.
http://www.millipore.com/catalogue/item/SCR021?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCR022?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/EV262?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/MIC230?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/M04S-03-5PK?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCM009?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/GF003?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/GF003?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCM009?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCR022?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCR021?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/M04S-03-5PK?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/MIC230?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/EV262?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/M04S-03-5PK?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/MIC230?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/EV262?cid=BIOS-C-EPDF-1007-1301-BS -
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Figure 3.
Brightfeld image o rat NSCs cultured in the CellASIC ONIX M04S Microuidic Plate onDay 8 (Let). Immunostaining o nestin (green) and Sox2 (red) indicated that these cellsremained undierentiated (Right).
Figure 2.
The microincubator controller provided precise control o gaseous microenvironment or themicrouidic cell culture. Gas exchange was accomplished within 15 minutes.
Automated immunostaining. 300 L PBS was
added to well 1 as the wash solution, 100 L o 4%
paraormaldehyde was added to well 2 as a fxing agent,
100 L o 0.2% BSA, 0.1% TritonX-100 in 1x PBS was
added to well 3 or permeation and blocking, 150 L o
primary antibody solution with 1% BSA was added to
well 4, and 100 L o secondary antibody was added to
well 5. To conduct the immunostaining automatically,
the pressure applied to each valve was programmedaccording to the ollowing parameters: V2 (fxing),
4 psi, 12 minutes; V1 (washing), 0.25 psi, 8 minutes;
V3 (permeation and blocking), 4 psi, 12 minutes; V1
(washing), 0.25 psi, 8 minutes; V4 (primary antibody),
4 psi, 60 minutes; V1 (washing), 0.25 psi, 8 minutes; V5
(secondary antibody), 4 psi, 40 minutes; V1 (washing),
0.25 psi, 5 minutes.
Testing NSC behavior with respect to gaseous
microenvironment. First, we determined the gaseous
microenvironment exchange rate. We purged the devicein pure nitrogen and let it stabilize or over 24 hours. A
pre-calibrated 5% CO2 gas was then introduced through
the microincubator. As depicted in Figure 2, the gas
exchanged thoroughly in about 15 minutes. To
investigate the impact o oxygen microenvironment on
NSC behavior, we seeded the cells under the normoxia
(20% oxygen) condition and let them stabilize in the
incubator or 24 hours. We then transerred the NSCs
onto three dierent microincubators and tri-gas mixers
tuned to three gas compositions buered with 5% CO2
and nitrogen: severe hypoxia (~0.1% O2), mild hypoxia
(~3% O2) and normoxia (~20% O2). Loading 300 L o the
culture medium in well 2, 3, 4 and 5, we were able
to culture the rat NSCs or up to our days, uninterrupted,
through pneumatic pumping o medium across the cell
culture chamber. Bright feld images were acquired every
day with a subset o cells immunostained or nestin
and Sox2.
ResultsThe key eatures o neural stem cells include sel renewal
and lack o dierentiation. To examine these signatures in
a gravity-driven perusion microuidic system over one
week, we used automated immunostaining to detect the
NSC markers, nestin and Sox2 (Figure 3). All the cells in the
chamber were successully stained, showing nestin in the
cytosol and Sox2 in the nucleus, which confrmed that the
rat NSCs maintained an undierentiated phenotype while
being cultured in this microuidic device.
6
5
4
3
%CO2
Time (minutes)
2
1
0
0:00:00 0:15:00 0:30:00 0:45:00 1:00:00 1:15:00 1:30:00 1:45:00
-
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Figure 4.
Rat NSCs cultured at low densities under mildlyhypoxic conditions (top) and under normoxicconditions (bottom). Under hypoxic conditions,the cells seemed to disaggregate and spreadinto discrete cell bodies while under normoxicconditions, the cells tended to aggregate.
Figure 5.
High density seeding o NSCs in a microuidicchamber under conditions o mild hypoxia causesormation o neurospheres (top row, inset boxat 72 hours). Neurospheres were also imaged at
higher magnifcation (20X, bottom let). Whenstained or nestin and Sox2 (bottom right), onlythe outer layers o the neurospheres showed bothnestin (green) and Sox2 (red) expression; thecore displayed bright spots o Sox2 expression.This pattern was possibly caused by neurospherecompression by the ceiling o the microuidicchamber.
We ound that cells detached and washed away, presumably
due to cell death, 24 hours ater exposure to severe hypoxia
(data not shown). At low cell density, cells disaggregated
under mildly hypoxic conditions, which promoted single-
layer cellular growth. In contrast, under normoxic conditions,
the cells aggregated into multilayer cellular masses (Figure 4).
Due to the cell loading variations, the initial seeding density
(0 hour) in the normoxia chamber was higher than the mild
hypoxia chamber (data not shown).
To urther explore the eect o microchamber culture o rat
NSCs, we seeded the cells at high density under mildly
hypoxic conditions (3% O2). Ater our days, the cells ormed
neurospheres, though the spheres were compressed by being
confned by the ceiling o the microchamber. Staining or
nestin and Sox2 revealed that the core o this tightly packed
mass contained only bright spots o Sox2 while the outer
ring o the neurospheres exhibited both nestin and Sox2
expression (Figure 5).
24 hours 48 hours 72 hours
24 hours
3% 02
20% 02
48 hours 40X objective
-
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Reerences1. Clarke, D. et al. Generalized Potential o Adult Neural Stem
Cells. Science 2000; 288 (5471): 1660-63.
2. Temple, S. Division and dierentiation o isolated CNS blast
cells in microculture. Nature 1989; 340: 471-73.
3. Serra M et al. J Neurosci Res. 2007 Dec;85(16):3557-66.
4. Lee YB et al. Exp Neurol. 2010 Jun;223(2):645-52.
5. Chung BG et al. Lab Chip. 2005 Apr;5(4):401-6.
6. EMD Millipore, User Guide: CellASIC ONIX M04S
Microluidic Plates. EMD Millipore Corporation, Billerica, MA
USA, 2012. Product Literature No. 00003670, Rev. A.
7. De Filippis L and Delia D. Hypoxia in the regulation o neural
stem cells. Cell Mol Lie Sci. 2011 Sep;68(17):2831-44.
8. Santilli G, et al. Mild hypoxia enhances prolieration and
multipotency o human neural stem cells. PLoS One 2009;
5(1):e8575
9. Takahashi K et al. Induction o pluripotent stem cells
rom adult human ibroblasts by deined actors. Cell 2007;
131(5):86187210. Storch A et al. Long-term prolieration and dopaminergic
dierentiation o human mesencephalic neural precursor
cells. Exp Neurol 2001; 170(2):317325
11. Aguirre A et al. Notch and EGFR pathway interaction
regulates neural stem cell number and sel-renewal. Nature.
2010 Sep 16;467(7313):323-7
12. Bakhru S et al. Direct and cell signaling-based, geometry-
induced neuronal dierentiation o neural stem cells.
Integr Biol (Camb). 2011 Dec;3(12):1207-14..
DiscussionThe cellular microenvironment, and the oxygen
microenvironment in particular, is known to regulate neural
stem cell metabolism, prolieration, surivival and ate7. Mild
hypoxia is a very physiologically relevant condition, given
that the oxygen concentration in most tissues is lower than
20%, which is the atmospheric oxygen concentration
(normoxia). Given that neural stem and progenitor cellshave been reported to show increased prolieration and
sel-renewal under mild hypoxic conditions8-10, studying the
eect o NSCs with respect to gaseous microenvironment
has the potential to advance research into the development
o NSC-based therapies or neurodegeneration.
We have successully developed the protocols to perorm
live cell imaging o rat NSCs using the CellASIC ONIX
Microuidic Platorm. By attempting various combinations
o these microenvironment parameters, we ound that the
rat neural stem cells exhibited dierent morphologies and
prolierated best under physiologic, mildly hypoxic
conditions, as reported in the literature8-10. In addition,
through the combination o high seeding density at
1 x 106/mL, polyornithine/laminin coating, continuous
perusion at 5 L/hour, and 3% oxygen (mild hypoxia) gas
microenvironment, the rat NSCs ormed a 3 mm x 3 mm x
0.1 mm neurosphere in 96 hours.
We also investigated the eect o varying cell density on
NSC dierentiation. The importance o cell density on the
dierentiation o NSCs has been recently established by
studies showing that NSC dierentiation can be regulated bycell-cell signaling, possibly through the epidermal growth
actor (EGF) and Notch signaling pathways11,12. Through live
cell imaging on a uorescent microscope, we discovered that,
while the peripheral cells around the neurosphere were
successully immunostained or two pluripotency markers,
nestin and Sox2, the image o the neurosphere itsel showed
condensed bright spots o Sox2 but no nestin, suggesting
that increased cell-cell contacts within the neurosphere may
aect NSC dierentiation.
In summary, we have demonstrated the combinatorial eect
o microenvironment parameters on rat NSCs and thecapability o imaging them with uorescent microscopy or
urther analysis. The platorm promises to acilitate assay
development or NSCs and provides a better-controlled
in vitromodel system or neurogenesis and neural
development research.
Available romwww.millipore.com.FEATURED PRODUCTS
Description Catalogue No.
CellASIC ONIX Microuidic System Package includes CellASIC ONIX Microuidic System, Maniold, Accessory
Box, and CellASIC ONIX FG Sotware
EV262
CellASIC ONIX Microincubator Package or Temperature and Gas Control: Includes CellASIC ONIX
Microincubator Controller, Microincubator Maniold, and Accessory Box
MIC230
M04S Microuidic Switching Plate or Mammalian Cells (4 Chambers) M04S-03-5PK
Adult Rat Hippocampal Neural Stem Cell Kit SCR021
Adult Rat Hippocampal Neural Stem Cells SCR022
Rat Neural Stem Cell Expansion Medium SCM009
Fibroblast Growth Factor basic, human recombinant GF003
http://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/EV262?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/MIC230?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/M04S-03-5PK?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCR021?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCR022?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCM009?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/GF003?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/GF003?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCM009?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCR022?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCR021?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/M04S-03-5PK?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/MIC230?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/EV262?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/M04S-03-5PK?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/MIC230?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/EV262?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BS -
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IntroductionCellular dierentiation is a undamental process in
developmental biology. Progenitor cells must have
the ability to dierentiate into more specialized cell
types or the body to respond to inections, eliciting
an immune response. Murine CD4+ T cells can give
rise to a variety o eector T, or T helper, cell subsets
depending on the nature o the immune response, and
subsequently release a distinct subset o cytokines.
During T cell dierentiation, a group o T helper (Th)
cell subsets play a critical role in both the innate and
adaptive immune response repertoire, deending the
body against oreign pathogens.
There are fve main subsets o dierentiated CD4+ T
cells: Th1, Th2, Th17, Treg, and Th cells. Each subtype
expresses a signature set o cytokines or transcription
actors that directs the immune response and regulates
dierentiation (Figure 1, page 2). For example, Th1
CD4+ T cells are important or protecting against
intracellular bacteria, ungi, and viruses, as well as being
involved in some autoimmune responses. These cells
mediate immune responses to intracellular pathogens
by producing intereron gamma (IFN-), and through
the secretion o IFN-, Th1 cells activate macrophages,natural killer (NK) cells, and CD8+ T cells. Unlike the
Th1 response, Th2 CD4+ T cells are oten associated
with humoral responses during which high levels o
Assessing enriched murine CD4+T cells dierentiated towardseector T helper cell lineages
with the Scepter 2.0 handheldautomated cell counterMark Santos, Wenying Zhang and Matthew Hsu
EMD Millipore Corporation
pathogen-specifc immunoglobulins are generated
to neutralize oreign organisms. Th17 cells play an
important role in the induction and propagation o
autoimmunity. Th17 cells signature cytokine is IL-17,
and IL-17 expression has been associated with many
autoimmune diseases such as multiple sclerosis,
rheumatoid arthritis, psoriasis, inammatory bowel
disease (IBD), as well as in allergic responses.
As CD4+ T cells dierentiate towards a specifc eector
T cell lineage, the cells dramatically change in size, a
unique physical hallmark o this dierentiation process.
We hypothesized that using the Scepter cell counter torapidly assess size distributions o cellular populations
would provide a quick, simple method or tracking T cell
dierentiation.
The Scepter cell counter captures the ease o
automated instrumentation and accuracy o impedance-
based counting using the Coulter principle in an
aordable, handheld ormat. The instrumentation has
been collapsed into a device the size o a pipette, and
uses a combination o analog and digital hardware or
sensing, signal processing, data storage, and graphicaldisplay in the orm o a histogram. The histogram output
provides a quick snapshot o cell size and density.
-
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Materials and MethodsEnriched CD4+ T cell isolation
Mouse spleens were harvested rom 8-10 week old
C57BL/6 mice (Charles River Laboratories) and prepared
according to the standard guidelines or obtaining single
cell suspensions (splenocyte harvesting). Homogenized
spleens were passed through a nylon mesh to ensure
single cell suspensions, ollowed by treatment with redblood cell lysing buer (Sigma, Catalogue No. R7757) to
remove red blood cells rom the culture. Cells were gently
resuspended in Hanks Balanced Salt Solution (HBSS;
Sigma, Catalogue No. H9394) and counted to determine
splenocyte recovery, and then applied to a CD4 isolation
column (R&D Systems, Catalogue No. MCD4C-1000) to
enrich or CD4+ T cells.
CD4+ T cell dierentiation towards specifc
eector T cell lineages
Mouse CD4+ splenocytes were dierentiated to
one o three distinct lineages through a process o
activation, expansion, and re-stimulation. The cells
were activated by culturing on anti-CD3 coated plates
with the single addition o a combination o growth
actors and antibodies in precise amounts to begin the
dierentiation process (EMD Millipore, Catalogue No.
FCIM025161 or Th1 cells; EMD Millipore, Catalogue No.
FCIM025162 or Th2 cells; EMD Millipore, Catalogue No.
FCIM025163 or Th17 cells). Cells were urther expanded
and then re-stimulated at days 4 and 6, respectively.
Ater day 6, the dierentiated Th cell cultures were
secreting their signature cytokines.
Scepter cell counting
The Scepter cell counter was used to count samples
ollowing the detailed on-screen instructions or each
step o the counting process. Briey, the user attaches
a 40 m sensor tip, depresses the plunger, submerges
the sensor into the sample, then releases the plunger
drawing 50 L o cell suspension into the sensor. The
Scepter detects each cell passing through the sensors
aperture, calculates cell concentration, and displays a
size-based histogram as a unction o cell diameter or
volume on its screen. Scepter 2.1 sotware was then
used to upload test fles rom the device and perorm
subsequent data analysis to determine cell sizes or
mouse CD4+ T cell (undierentiated) versus Th1, Th2, or
Th17 eector T cell cultures (dierentiated).
In this study, we demonstrate a method or driving
murine CD4+ T cell dierentiation towards Th1, Th2,
or Th17 cells in vitroand measuring cell size changes
beore and ater this process using the Scepter cell
counter. We have employed Scepter technology or cell
volume/size determination to investigate the relationship
between cell dierentiation and cell size changes. We
confrmed T cell dierentiation status by using a ow
cytometry assay to measure intracellular cytokine
production or Th1, Th2, and Th17 cell types. Although
the Scepter cell counter was intended primarily as a
cell counting device, here we demonstrate how this cell
counter can also unction as a reliable tool or other
diverse biological applications.
Figure 1.
Eector CD4+ T cell lineage commitment. CD4 + T cellscan give rise to many subtypes depending on the type orequired immune response. A complex process includingactivation o T cell receptor, along with a local cytokineenvironment, polarize CD4+ T-cells to a defned lineage o
mature cytokine-producing helper T-cells and Treg cells.
DC
IL-12R
TH1
T-bet
STAT4
STAT1
IL-2R
TRE G
Foxp3
STAT5
IL-21R
TFHTH
Bcl-6
STAT3
IL-4R
TH2
GATA3
STAT6
STAT5a
IL-23R
TH17
RORt
STAT3
IL-12
CD80
CD28
TCR
CD28CD85
pMHCII
IFN
IL-18
IL-2
IL-4
IL-33
IL-6
IFN
IL-2
LT
IL-21
IL-6
TGFIL-23
TGF
IL-2
IL-4
IL-5
IL-13
IL-25
IL-17
IL-17F
IL-22
IL-21
IL-21
IL-17
TGF
IL-10
IL-35
naive
http://www.millipore.com/catalogue/item/FCIM025161?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025162?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025163?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025163?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025162?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025161?cid=BIOS-C-EPDF-1007-1301-BS -
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Evaluation o Th1, Th2, and Th17 cell lineages
by ow cytometry
All ow cytometry assays were perormed using EMD
Millipores FlowCellect Mouse Th intracellular cytokine
kits (EMD Millipore, Catalogue No. FCIM025123 or Th1
cells; EMD Millipore, Catalogue No. FCIM025124 or Th2
cells; EMD Millipore, Catalogue No. FCIM025125 or Th17
cells). All reagents and detailed instructions are provided
in the user guides or each kit. Please reer to each kits
specifc instruction or more detailed inormation. All
sample acquisition and data analysis was perormed
using the guava easyCyte system (EMD Millipore,
Catalogue No. 0500-4008).
Comparison o Scepter cell analysis with an
automated cell viability image-based system
Using the Scepter cell counter, cell sizes o CD4+ T cells
(undierentiated) and Th1 CD4+ T cells (dierentiated)
were analyzed in triplicate assays (three independent
experiments, plus three samples per experiment) andstandard deviation values were determined. In parallel,
both sets o samples were analyzed using a more
expensive automated cell viability analyzer.
ResultsMouse CD4+ T cells were treated with established
lineage-specifc actors or six days to drive
dierentiation towards eector T cell-specifc lineages:
Th1, Th2, or Th17 cells. Using the EMD Millipore mouse
dierentiation tool kits as described previously, we were
able to successully achieve cell dierentiation in an invitrosystem, and these results are captured using the
Scepter device based on cell size discrimination. Cross
validation studies were also perormed by ow cytometry
using EMD Millipores mouse intracellular cytokine kits to
veriy and confrm our results.
Scepter analysis o CD4+ T cell dierentiation
towards the Th1 lineage
Beore induction, undierentiated CD4+ T cells were
measured using the Scepter producing a mean cell
diameter o 6.4 m (Figure 2, blue histogram). Ater
six days in culture under the inuence o Th1 lineage-
inducing reagents, such as lineage specifc cytokines and
growth actors, resulting Th1 cells were measured using
the Scepter cell counter. The mean cell diameter o
this dierentiated cell population was 10.4 m (Figure
2, red histogram). Figure 3 shows brightfeld microscopy
analysis o the same cells, confrming that their average
diameter had indeed increased.
Figure 2.
Accurate discrimination between mouse CD4+ T cells(undierentiated) and dierentiated Th1 cells based oncell size. Using a 40 m sensor, the Scepter cell counterenabled the discrimination o cell types based on size withhigh resolution. Mouse CD4+ T cells were measured beoreand ater dierentiation towards the Th1 cell lineage. Ater sixdays in culture both cell types were measured and recorded.As indicated by the Scepter histogram data, cells increased insize rom 6 to 10 m, on average.
Figure 3.
Microscopy images beore and ater T cell dierentiation:confrmation that cell size is accurately measured by theScepter cell counter. CD4+ T cells prior to treatment underdierentiation conditions exhibited cell diameters around5-6 m, as shown in (A). In (B), six days ater exposure tospecifc cytokines and growth actors to induce dierentiationtowards the Th1 cell lineage in vitro, cells appear much largerwhen visualized at the same magnifcation.
A. Day One (1): Activation B. Day Six (6): Re-stimulation
400
320
240
Count
160
80
03 6
Diameter (m)
9 12 15 18
CD4+ T cells (Undifferentiated)
Th1 CD4+ T cells (Differentiated)
http://www.millipore.com/catalogue/item/FCIM025123?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025124?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025125?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/0500-4008?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025125?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025124?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025123?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/0500-4008?cid=BIOS-C-EPDF-1007-1301-BS -
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Figure 4.
Flow cytometry validates the dierentiationtowards Th1 cells, confrming the resultsobtained using the Scepter cell counter. Usingthe FlowCellect Mouse Th1 Intracellular CytokineKit (Catalogue No. FCIM025123), percentages odierentiated versus undierentiated CD4+ T cellswere determined. Undierentiated cells did notshow any signifcant expression o the signaturecytokine IFN-as seen in (A), however, ater sixdays in culture under lineage specifc conditionstoward Th1 cells, cells showed positive expressiono IFN-as seen in (B).
Validation o Th1 cell dierentiation
using ow cytometry
In order to validate that the cell types measured were
truly dierentiated cells, a ow cytometry assay was
perormed to measure intracellular cytokine production.
As shown in Figure 4, 91.2% o the day 6-stimulated
cell population expressed IFN-, a characteristic Th1
cell marker, confrming that the increase in cell size was
indeed a phenotype o dierentiated Th1 cells as studiedin Figures 2 and 3 (page 3).
The Scepter cell counter precisely measured cell
diameter changes across multiple samples (Table 1,
below). Moreover, the data showed that the Scepter
cell counter could achieve similar or more precise
measurements when compared to an alternative
benchtop instrument.
Table 1.
Scepter cell counter versus the automated cell viability image-based system: measuring cell size diameter or both naive CD4+ T cells (undierentiated)and Th1 CD4+ T cells (dierentiated).
Experiment #1 Experiment #2 Experiment #3
n=1 n=2 n=3 n=1 n=2 n=3 n=1 n=2 n=3
Average
diameter
(m)
Standard
Deviation
Scepter Values
Day 1
(Undierentiated)
6.33 6.36 6.4 6.14 6.24 6.66 6.33 6.34 6.43 6.36 0.14
Day 6
(Dierentiated)
11.0 11.02 11.07 10.05 10.05 10.28 9.97 9.97 9.98 10.38 0.50
Automated Cell Viability image-based System
Day 1
(Undierentiated)
7.01 6.48 6.87 6.49 6.57 6.91 6.41 6.88 6.92 6.73 0.23
Day 6
(Dierentiated)
11.31 11.40 11.88 10.25 10.37 10.79 10.02 10.13 10.28 10.71 0.66
104
103
102
101
100
100 101 102
CD4 (RED-HLog)
IFN(
YEL-HLog)
103 104
104
103
102
101
100
100 101 102
CD4 (RED-HLog)
IFN(
YEL-HLog)
103 104
A. Day One (1): Activation
B. Day Six (6): Re-stimulation
http://www.millipore.com/catalogue/item/FCIM025123?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025123?cid=BIOS-C-EPDF-1007-1301-BS -
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400
320
240Count
160
80
0
3 6Diameter (m)9 12 15 18
CD4+ T cells (Undifferentiated)
Th2 CD4+ T cells (Differentiated)
450
350
250Count
150
50
03 6
Diameter (m)
9 12 15 18
CD4+ T cells (Undifferentiated)
Th17 CD4+ T cells (Dif ferentiated)
Figure 6.
Flow cytometry confrmation odierentiation o enriched CD4+ T cellsto Th2 and Th17 cells. Cell types which
produced larger cell diameters as detectedby the Scepter cell counter were the samecells cultured or six days as shown in (B) and(D), whereas their progenitor cell types areshown in (A) and (C). Only cells in (B) and (D)expressed the signature cytokines IL-4 andIL-17 or Th2 and Th17 cells, respectively.
Figure 5.
CD4+ T cell dierentiationtoward Th2 and Th17cell lineages can beclearly identifed usingScepter based on cellsize. Using a 40 m sensor,the Scepter cell counterenables the discriminationo cell types based on sizewith high resolution. Thesize distributions o Th2and Th17 cell populationswere compared to the sizedistribution o the CD4+ Tcell progenitor cell type. BothTh2 and Th17 cells graduallyincreased in size rom 6to 10 m ater six days odierentiation.
CD4+ T cell dierentiation towards
the Th2 and Th17 lineages
CD4+ T cells were induced to dierentiate towards
both Th2 and Th17 cell lineages using EMD Millipores
Mouse Th Dierentiation Tool kits or Th2 and Th17
cells (Catalogue No. FCIM025162 and FCIM025163,
respectively). Beore induction, undierentiated CD4+ T
cells were measured using the Scepter cell counter as
having a mean cell diameter o 6.3 m (Figure 5, blue
histogram). Ater six days in culture under the inuence
o lineage-specifc reagents, cells were dierentiated
towards the Th2 and Th17 cell types. The resulting cell
populations were then measured using the Scepter cell
counter, which reported mean cell diameters o 10.1 m
and 9.8 m or Th2 and Th17 cells, respectively (Figure 5,
red histogram).
In order to validate that the cell types measured were
truly dierentiated cells and specifc or each cell
type described, we used ow cytometry to measure
intracellular cytokine production or the particularcell type in question. As shown in Figure 6, increased
expression o IL-4 or Th2 cells and increased
expression o IL-17 or Th17 cells confrmed that the
observed increases in cell size were true phenotypes o
dierentiated cells.
As with Th1 cell dierentiation, the Scepter cell counter
precisely measured cell diameter changes across multiple
samples o progenitor T cells and those dierentiated
toward Th2 and Th17 lineages (Table 2). Again, our data
showed that the Scepter cell counter could achievemore precise measurements when compared to an
alternative benchtop instrument.
A. Undierentiated CD4+ T cells versus Th2 cells
B. Undierentiated CD4+ T cells versus Th17 cells
104
103
102
101
100
100 101 102
CD4 (RED-HLog)
IL-4
(YEL-HLog)
103 104
104
103
102
101
100
100 101 102
CD4 (RED-HLog)
IL-4
(YEL-HLog)
103 104
104
103
102
101
100
100 101 102
CD4 (RED-HLog)
IL-17
(YEL-HLog)
103 104
104
103
102
101
100
100 101 102
CD4 (RED-HLog)
IL-17
(YEL-HLog)
103 104
http://www.millipore.com/catalogue/item/FCIM025162?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025163?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025163?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025162?cid=BIOS-C-EPDF-1007-1301-BS -
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Table 2.
Scepter cell counter versus the automated cell viability image-based system: Measuring cell size diameter or naive CD4+T cells (undierentiated), Th2 and Th17 CD4+ T cells (dierentiated). Data points with smaller standard deviations clearlydemonstrated the precision o the Scepter cell counter when compared to a more expensive cell counting instrument.
Table 1 (page 4) and Table 2 (below) were summarized in a bar graph showing the clear phenotypic dierence between
undierentiated and dierentiated CD4+ T cell populations, underscoring the utility o the Scepter cell counter to
provide a rapid, simple, accurate method or tracking immune response.
Th2 cell lineage
Th17 cell lineage
Experiment #1 Experiment #2 Experiment #3
n=1 n=2 n=3 n=1 n=2 n=3 n=1 n=2 n=3
Average
diameter
(m)
Standard
Deviation
Scepter Values
Day 1
(Undierentiated)
6.24 6.26 6.30 6.37 6.37 6.38 6.40 6.42 6.43 6.35 0.07
Day 6
(Dierentiated)
10.07 10.11 10.24 9.92 10.05 10.11 9.88 9.97 10.27 10.07 0.13
Automated Cell Viability image-based System
Day 1
(Undierentiated)
7.00 7.18 7.20 7.01 7.15 7.33 7.35 7.47 7.59 7.25 0.20
Day 6
(Dierentiated)
10.68 10.95 10.99 11.04 11.10 11.48 10.57 10.98 11.36 11.02 0.29
Experiment #1 Experiment #2 Experiment #3
n=1 n=2 n=3 n=1 n=2 n=3 n=1 n=2 n=3
Average
diameter
(m)
Standard
Deviation
Scepter Values
Day 1
(Undierentiated)
6.24 6.26 6.30 6.37 6.37 6.38 6.40 6.42 6.43 6.35 0.07
Day 6
(Dierentiated)
9.69 9.83 9.83 9.73 9.81 9.96 9.74 9.81 9.84 9.80 0.08
Automated Cell Viability image-based System
Day 1
(Undierentiated)
7.00 7.18 7.20 7.01 7.15 7.33 7.35 7.47 7.59 7.25 0.20
Day 6
(Dierentiated)
10.20 10.51 10.69 10.54 10.72 10.82 10.35 10.50 10.87 10.58 0.22
-
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ConclusionsT cell dierentiation and its role in the immune response
are integral or both innate and adaptive immunity. The
importance o eector T cells and their ability to combat
against various types o pathogens, as well as maintaining
immune homeostasis, should never be underestimated.
In order to stay healthy, the body specifcally recruits CD4
eector T cells to execute its immune unctions. Dysregulated
Th cell unction oten leads to inefcient clearance o
pathogens, which can cause diseases o inammation and
impact autoimmunity.
Dierentiation o CD4+ T cells into ully unctional eector
T cells is characterized by both the production o signature
cytokines as well as a concomitant increase in cell size.
As demonstrated in this study, Th1, Th2, and Th17 cells
expanded rom approximately 6 to 10 m when compared
to the progenitor CD4+ T cell type. This expansion upon
dierentiation was clearly, accurately, and precisely measured
using the Scepter cell counter.
The Scepter provides a rapid, easy, and inexpensive method
or assessing naive CD4+ T cell dierentiation toward specifc
eector T cell lineages. This handheld, automated cell counter
delivers precise, reliable cell size measurements, which
can provide the researcher with a quick snapshot o the
dierentiation status and, ultimately, immune response.
Figure 7.
Cell diameter change during CD4+ T cell dierentiation(toward Th1 / Th2 / Th17 cell lineages) as measured by theScepter cell counter.
Reerences1. Wan, Y.Y., et. al. How diverse CD4 eector T cells and theirunctions. J Mol Cell Biol. 2009; 1(1): 2036.
2. Zhu, J., et. al. Dierentiation o eector CD4 T cell
populations. Annu Rev Immunol.2010; 28:445-89.
3. Zhou, L., et. al. Plasticity o CD4+ T cell lineage
dierentiation. Immunity 2009; 30(5):646-55.
4. Fietta, P., et. al. The eector T helper cell triade. Riv
Biol.2009;102(1):61-74.
Mouse CD4+ T cell Diameter (m)
0CD4+ T cells Th1
Differentiated(Day 6)
Undifferentiated(Day 1)
Th2 Th17
2
4
6
8
10
12
-
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Reagents/Kits
Description Qty/Pk Catalogue No.
Scepter 2.0 Handheld Automated Cell Counter
with 40 m Scepter Sensors (50 Pack) 1 PHCC20040
with 60 m Scepter Sensors (50 Pack) 1 PHCC20060
Includes:Scepter Cell Counter 1
Downloadable Scepter Sotware 1
O-Rings 2
Scepter Test Beads 1 PHCCBEADS
Scepter USB Cable 1 PHCCCABLE
Scepter Sensors, 60 m 50 PHCC60050
500 PHCC60500
Scepter Sensors, 40 m 50 PHCC40050
500 PHCC40500
Universal Power Adapters 1 PHCCP0WER
Scepter O-Ring Kit, includes 2 O-rings and 1 flter cover 1 PHCC0CLIPGuava easyCyte 8HT Flow Cytometer 1 055-4008
Description Catalogue No.
FlowCellect Mouse Th1 Dierentiation Tool Kit FCIM025161
FlowCellect Mouse Th2 Dierentiation Tool Kit FCIM025162
FlowCellect Mouse Th17 Dierentiation Tool Kit FCIM025163
FlowCellect Mouse Th1 Intracellular Cytokine Kit FCIM025123
FlowCellect Mouse Th2 Intracellular Cytokine Kit FCIM025124
FlowCellect Mouse Th17 Intracellular Cytokine Kit FCIM025125
Available romwww.millipore.com.
FEATURED PRODUCTS
http://www.millipore.com/catalogue/item/PHCC20040?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC20060?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCCBEADS?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCCCABLE?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC60050?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC60500?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC40050?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC40500?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCCP0WER?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC0CLIP?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/055-4008?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025161?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025162?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025163?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025123?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025124?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025125?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025125?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025124?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025123?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025163?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025162?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025161?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/055-4008?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC0CLIP?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCCP0WER?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC40500?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC40050?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC60500?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC60050?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCCCABLE?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCCBEADS?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC20060?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC20040?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BS -
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Product Review
Autophagy:Mechanisms and connections
to apoptosisChandra Mohan, Ph.D., EMD Millipore Corporation
Phagophore
LC3
LC3Autophagosome Autolysosome
AutophagyInduction
Lysosome
Low NutrientsExerciseAdiponectinGhrelin
LKB1
CAMKK2
GrowthFactors
Ulk1/2
ProteinAggregate
ProteinAggregate
Ulk1/2
FIP200
Agt13
mTORmTORC1
AKT
AMPK
P
P P
P
P P
Autophagy is a highly regulated homeostatic
degradative process in which cells destroy and recycle
their own components via the lysosomal machinery.
In mammalian cells, autophagy is believed to occur
constitutively at basal rates. Under conditions o
extreme starvation, cells use this process to reallocatenutrients rom less important processes to more
essential processes required or survival. However, i
cellular damage becomes irreparable, cells can destroy
themselves completely by autophagy. Several processes
can be classiied under the general term autophagy.
Figure 1.
Steps o macroautophagy include induction, phagophoreormation, autophagosome maturation and autophagosome-
lysosome usion. LC3 is a small protein that is targeted tothe phagophore membrane by a ubiquitin ligase-like enzymecascade. LC3 mediates phagophore elongation to orm themature autophagosome.
However, one common element o autophagy pathways
involves the importation o cytoplasmic components
into the lysosome. In eukaryotes, autophagy unctions
solely as a degradative and remodeling pathway, while
in yeasts, autophagy also plays a role in biosynthesis.
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Three types o autophagyGenerally, three types o autophagy have been
recognized. They are chaperone-mediated autophagy,
microautophagy, and macroautophagy. Chaperone-
mediated autophagy involves the direct translocation
o cytosolic proteins across the lysosomal membrane.
It requires cytosolic and lysosomal chaperones to
unold substrates. During microautophagy, cytoplasmis sequestered directly at the lysosomal surace by
separation and/or invagination o the lysosomal
membrane. In macroautophagy the sequestering o
membrane is distinct rom lysosome and it involves the
ormation o autophagosome that uses with lysosome,
which provides the hydrolytic enzyme machinery.
The used structure is termed as autophagolysosome.
The maturation o autophagolysosome requires
acidiication by H1-ATPase. Hence, several inhibitors
o H1-ATPase, such as Bailomycin A1, are shown
to diminish autophagy. Following their breakdown
macromolecules are released back into the cytoplasm
or reuse in the metabolic processes.
Steps o macroautophagyThe process o autophagy can be divided into several
phases. In the initial phase, the cell senses signals
released in response to lack o nutrients, hypoxia,
hormones or other sources o cell stress. These signals
can induce macroautophagy, either via inhibition
o mTOR or by AMPK activation o the ULK1/2kinase complex (Atg1 in yeast). mTOR is essential to
nutrient-sensing signal transduction and regulation o