Approaches to Cell Proliferation and Cell Counting.
Mike Ignatius, Ph.D.
Product Manager, Molecular Probes/Invitrogen
Ed Leber,
Technical Service Rep on e-Chat
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Technologies Invitrogen Offers.
• Cell Number by Metabolic Indicators– Vybrant Cell Metabolism Assay Kit– LIVE/DEAD Calcein AM Vitality Kits– Luminescent: ATP Determination Kit– Colorimetric: Alamar Blue, MTT
• Cell Number by DNA Content– CyQuant™, CyQuant™ NF Cell Division
by DNA synthesis– BrdU, antiBrdU– Click-iT™ EDU
• Cell Division by Tracer Dye Analysis– CFSE (CellTrace kits and reagent)– Vybrant™ DiI
• Cell Division by Cell Cycle Analysis– DyeCycle™ Dyes
• Other Solutions– Counting Beads.
• Countess, Automated Hemocytomer Like Cell Counting
Let me count the ways….
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Covered Today
• Cell Number by Metabolic Indicators– Vybrant Cell Metabolism Assay Kit– LIVE/DEAD Calcein AM Vitality Kits– Luminescent: ATP Determination Kit– Colorimetric: Alamar Blue, MTT
• Cell Number by DNA Content– CyQuant™, CyQuant™ NF, CyQuant Direct™
Cell Division by DNA synthesis– BrdU, antiBrdU– Click-iT™ ( EDU
• Cell Division by Tracer Dye Analysis– CFSE (CellTrace kits and reagent)– Vybrant DiI
• Cell Division by Cell Cycle Analysis– DyeCycle Dyes
• Other Solutions– Counting Beads.
• Countess®, Automated Hemocytomer Like Cell Counting
Different assays
give different answers
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Reagent summary for cytotoxicity or proliferation analysis
Extremely rapid
Easy Edu replaces difficult BrdU assays
Samples remain intact
Imaging, HCS, flow and microplate formats
No wash assay
Highly accurate, sensitive, and linear
Workflow choices
Easy, HTP, homogeneous assay
Economical
Non toxic to cells
Why choose:
Direct indicator of cell proliferation
Cell counting via DNA content to assess cytotoxic or proliferative effects independent of cellular metabolism
Cell viability and cytotoxicity, & indirect proliferation measurement
Primarily used for:
Which cells are actively proliferating?
Have my cells multiplied? How many cells are present?
What is the metabolic health of the cells?
Answers the question:
Measures rate of new DNA synthesis by EdU nucleoside analog incorporation into DNA with detection by copper-catalyzed “click” reaction
Quantitates relative number of cells in a population based on total DNA content by measuring DNA binding dye
Metabolic assay for cellular reducing environment where readout is proportional to number of living cells
How it works:
Click-iT™ EdU KitsCyQuant® AssaysalamarBlue®
Product
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alamar Blue®- HTS Ready
• Measure of cell health thru cellular reducing potential• Live cells maintain reducing environment in cytosol• Excitation 540-570 nm / Emission 580-610 nm
– Monochrometer 560/590 nm• Simple add and read protocol• Proprietary reagent mix allows less volume addition and more dynamic range
Blue Red
resazurin resorufin
Healthy Cells
Dying/Dead Cells
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alamarBlue®
How does the assay work? • Cellular metabolism converts alamarBlue® from
blue to a pink & fluorescent product that is released into media
• Healthy cells are more metabolically active than sick or dead cells :
– Healthy cells convert more reagent from blue to pink
Protocol in brief:
1. Add reagent to cells (10% v/v)
2. Incubate at 37 C for 15-30 min
3. Read results by absorbance or fluorescence in microplate reader
4. Returned to incubator and use from other assays e.g. reporter gene studies
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alamarBlue® sensitivity
Sensitivity of alamarBlue® reagent after 18 hour incubation
The inset graph shows alamarBlue® to be linear over the range from 50 to 5,000 cells/well after 18 hour incubation of cells with reagent.
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alamarBlue® gives nearly identical results at a fraction of the cost of CellTiter® Glo
Comparison of alamarBlue® reagent with CellTiter-Glo®. HUVEC cells were treated with tamoxifen for 24 hours prior to performing the cytotoxicity assays. The alamarBlue® (AB) and CellTiter-Glo® (CTG) assays were performed according to the manufacturer’s instructions.
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alamarBlue® is more accurate in confluent cultures
alamarBlue® accurately shows no cell death in the confluent culture, whereas since ATP is depleted, CellTiter-Glo® indicates false cell death.
“Cells that become quiescent upon reaching confluency may have decreasing ATP levels and thus may not be accurately measured by the CellTiter® Glo assay at confluence.”
-0.50 -0.25 0.00 0.25 0.50 0.75 1.00 1.25 1.500
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alamarBlue®
CellTiter-Glo®
log Tamoxifen (μM)
Bkg
Sub
RFU
Bkg Sub R
LU
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CyQUANT® Kits
Most Sensitive and Robust Product Family for DNA-based Cell Proliferation and Cytotoxicity Studies
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CyQUANT® Family of Cell Proliferation Kits
• Highly Sensitive: Much more sensitive than metabolically based assays to small changes in cell population/number (4 log dynamic range).
• Easy to use: Compatible with automation and high throughput analysis.
• Convenient: Choose from one of three kits to suit your assay and work flow needs.
• Accurate: DNA is tightly regulated and signal is not dependent on metabolic state, growth conditions—no better HTS assay for cell number
• Robust: Highly reproducible and not prone to compound interference—from assay to assay and lab to lab.
Nucleic acid-specific dye that fluoresces strongly green (ex=488 nm) when bound to DNA
Only DNA binding reagents suitable for HTS
100 platesC35012CyQUANT® Direct Cell Proliferation Assay Kit
2 platesC35007CyQUANT® NF Cell Proliferation Assay Kit
10 platesC35006CyQUANT® NF Cell Proliferation Assay Kit
10 platesC35012CyQUANT® Direct Cell Proliferation Assay Kit
10 platesC7026CyQUANT® Cell Proliferation Assay Kit
SizeSKUName
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DNA Based Cell Number with CyQuant® Assays: Same Principle, Different Workflow Options.
First up, the original CyQUANT® assay (released in 1995)
1. Wash, Freeze, Add and Read. 2. Can be stored frozen for 4 weeks prior to read.3. Sensitive: from 50 to 250,000 cells4. Best in class dynamic range, linear over 4 logs5. Flexible: top- or bottom-read; 96 or 384 well formats.6. Ideal for time course studies (proliferation)
You can count on it!
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CyQuant® NF: No Freeze, cells intact, HCS ready!
Second, the CyQuant® NF assay (released in 2005)
1. No freeze means antigenicity and cellular structures are preserved2. Multiplex with antibody- or dye-based assays in flow or HCS3. Use on fixed cells—no dependence on metabolic activity4. Simple protocol: Wash, Add and Read. Stable for hours.5. Best in class DNA based proliferation assay.
NF = No Freeze, New Formulation, Nice Format
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Cyquant NF™ - Pharmacology
Normal Cells Toxicity DataTamoxifen 24 Hrs
Cyquant NF
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.251,000,000
3,000,000
5,000,000
7,000,000
9,000,000
11,000,000
13,000,000
IC50SH-SY5Y-59.2 μM
Tamoxifen (log M)
Bkg
Sub
RFU
Primary Cells Toxicity DataTamoxifen 24 Hrs
Cyquant NF
-0.50 -0.25 0.00 0.25 0.50 0.75 1.00 1.25 1.500
500,000
1,000,000
1,500,000
2,000,000
IC50HUVEC- 6.3 μM
Tamoxifen (log M)
Bkg
Sub
RFU
•Pharmacological findings match those seen with alamar Blue®•Primary Cells more sensitive to drug effects as seen with AB
•Unlike alamarBlue® a media removal step is required•Haven’t yet tested with New CyQuant™ DIRECT.•Mechanistic readout is not based on cellular metabolism
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alamarBlue® and CyQUANT® can be used together
HepG2 cells were incubated with tamoxifen for 24 hours prior to the addition of reagnets. The indicated IC50 values demonstrate that alamarBlue®reagent can be multiplexed with other cytotoxic indicators and still give accurate results.
Potential multiplexing advantages
•Save on resources
•Get a more accurate indication of cell health due to the multiple assessment modes used
A) alamarBlue® reagent
B) CyQUANT® NF
C) alamarBlue® reagent and CyQUANT® NF
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Now: CyQUANT® Direct Cell Proliferation Assay
Quantitative, sensitive, and non-radioactive indicator for cell cytotoxicity and proliferation
Dye turns fluorescent upon binding to DNA
Normal Cells Toxicity DataTamoxifen 24 Hrs
Cyquant NF
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.251,000,000
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IC50SH-SY5Y-59.2 μM
Tamoxifen (log M)
Bkg
Sub
RFU
Cells + Drug candidate
Add CyQUANT® reagent Measure
What is it?
Homogenous version of CyQUANT® dye, combined with a masking dye that prevents staining of cells with damaged membrane
Why use it?
• Convenient workflow – one addition, fast labeling, no washes, cell lysis or temperature equilibrations required. Room temp storage.
• Reported to be among the most sensitive measures of cytotoxicity and the most accurate assay for cell number (proliferation)
• Readout independent of metabolic state gives excellent robustness and may reduce the risk for false positives
• Highly robust and accurate
New!!!
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CyQUANT® Direct: Add and Read Protocol
30 to 60 minTreatment Signal from stained dead & damaged
cells blocked by masking dye
– Homogenous assay with fast, work flow-friendly protocol
– Only healthy cells are stained—two assays in one (DNA and membrane integrity)
– Metabolic activity is not needed for staining (e.g. confluent, quiescent cells)
– Can be used with either adherent or suspension cells (bottom read recommended)
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CyQUANT® Direct: Visualize the Results!
PrimaryHASMC Jurkat
Hep G2 CHO
Inspect cell morphology, confluence, cell numbers visuallyMultiplex with spectrally distinct fluorescent markers or
luminescence readoutInspect by microscope and analyze by HCS
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Choose CyQUANT® Kits Based on Application/Workflow
CyQUANT® NF Cell Proliferation Assay Kit1. No freeze, includes permeabilization step2. Antigenicity and cell structure preserved.3. Recommended for flow, HCS, imaging and assays using antibodies.
CyQUANT® Cell Proliferation Assay Kit1. Requires freeze step, but most sensitive2. Store frozen for weeks prior to read3. Linear over 4 logs from 20 to 50,000 cells4. Recommended for time course, batch assays and top-read instruments
CyQUANT® Direct Cell Proliferation Assay Kit1. Homogenous by combo of masking dye and DNA stain—add, mix, read 2. Fast and convenient—room temp storage, no lysis, no freeze, no perm 3. Most robust, accurate and stable signal4. Measures DNA content and membrane integrity (proliferation and
cytotoxicity)5. Recommended for high throughput applications in bottom read mode
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Viability AssaysLive Dead, Cytotoxicity Assay.In live cells, esterase converts Calcein AM from non-fluorescent to green. Dead cells accumulate the another dye to stain red.
•Red Dead, Green Growing.
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Viability & Vitality: LIVE/DEAD® Kits
No488nmPropidium IodideSYBR® 14 dyeLIVE/DEAD® Sperm Viability
KitL7011
Yes488nmGreen Dye
brightGreen Dye
dimLIVE/DEAD® Reduced
Biohazard Cell Viability Kit #4L23105
Yes488nmRed Dyebright
Red Dyedim
LIVE/DEAD® Reduced Biohazard Cell Viability Kit #3L23102
YesUVBlue Dyebright
Blue Dyedim
LIVE/DEAD® Reduced Biohazard Cell Viability Kit #2L23101
Yes488nmReactive RedSYTO® 10 dyeLIVE/DEAD® Reduced
Biohazard Cell Viability Kit #1L7013
No488nmPropidium IodideDiOC18(3)LIVE/DEAD® Cell-
Mediated/Cytotoxicity KitL7010
No488nmEthidium Homodimer-
1Calcein AM
LIVE/DEAD® Viability/ Cytotoxicity Kit
L3224
Fix?ExcitationDead StainLive StainKit NameSku #
LIVE/DEAD® lineup: pick your colors
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Click-iT™ Labeling and Detection
Replacing radioactivity with fluorescence for sugars, amino acids and nucleic acid analysis in living cells.
Novel tools for fluorescence imaging, gel electrophoresis, and flow cytometry - AND CELL PROLIFERATION – BRDU REPLACEMENT
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Concept introduce by K. Barry Sharpless in 2001. Has become associated in particular with one reaction.
Kolb HC, Finn MG, Sharpless K.B.; (2001) Angewandte Chemie International Edition V40:2004-2021.
Tornoe CW, Christensen C, Meldal M; (2002) J Org Chem. May 3;67(9):3057-64.
Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)
•Highly efficient•Rapid•Bio-orthogonal•Components are inert to biomolecules•No side reactions
DetectionProbe N3 Macromolecule
N NN
R''
R'
Macromolecule
DetectionProbe
Copper I
Just what is “click” chemistry?
+
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•Metabolic•Enzymatic•Chemical
Phosphoproteins
Glycoproteins
Total proteins
DNA/RNA
Lipids
Incorporate azide/alkyne tag into macromolecule of interest
•1D-2D gel imaging
•Western blotting
•Fluorescence microscopy
•Flow cytometry
•Enrichment
•In situ hybridization
•Fluorescent•Biotinylated•Affinity tags
Click-iT label macromolecule with desired azide/alkyne probes
The Click-iT™ 2-step labeling and detection approach
•REPLACEMENT FOR BrDU for PROLIFERATION
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Acid or DNase
Br
Br
Br
Br
BrdU - is inaccessible to the BrdU antibody
Requires DNA denaturation for strand separation
Cell cycle dye requires the DNA to be double-stranded
Difficult balancing act:
Numerous protocols: acid, heat, or nuclease for DNA denaturation
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Ethynyl dU Bromo dU
H
Nucleoside analogue structures
Click-analogue
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Chemistry
Click labeling doesn’trequire DNAdenaturation
Dye azide reacts with the alkyne on the double stranded DNA
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Click-iT™ EdU Protocol
TOTAL TIME70 minutes
Image
15 minutesWash 3X
15 minutesNuclear counterstain
10 minutesWash 2X
30 minutesClick-iT™ detection reaction
Incubate with EdU or BrdU, fix & permeabilize sample
With Click-iT™ EdU
• Measure proliferation in cells or tissue
• Time to complete: <2 hours
• Detect by fluorescence microscopy, flow cytometry or high-throughput imaging (HCS)
BrdU Protocol
15 minutesNuclear counterstain
15 minutesWash 3X
15 minutesWash 3X
15 minutesWash 3X
60 minutesBlock
1-16 hoursAnti-BrdU incubation
120 minutesSecondary antibody incubation
TOTAL TIME 6-21 hours
Image
15 minutesWash 3X
12 minutesNeutralize
40 minutesHCl Denaturation
15 minutesWash 3X
Click-iT™ EdU vs. BrdU Workflow
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Click-iT™ EdU vs BrdU Detection in Tissue Sections
•ClickiT™ or Ticket…
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Click-iT™ EdU in Adult Brain Tissue.
Adult Mouse brain section, an organ whose cells almost never divide.
A sole EdU-labeled cell can be easily identified on this brain section.
Courtesy of Adrian Salic, Harvard.
Research published in
Feb 2008, PNAS 105(7) 2415-2420.
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Muntjac cells pulsed with 10 μM EdU for 1 hour
Multicolor imaging with Click-iT™ EdU. Newly synthesized DNA with Click-iT™EdU (pink), tubulin (blue), golgi (green) and peroxisomes (orange).
EdUtubulingolgiperoxisome
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Cell cycleMitosis
S = period of EdU uptake
G0G1
G0: quiescent cells
G1: Period of cell growth where a cell has 2N nuclear DNA content (2 copies of nuclear genome)
S: Period where nuclear DNA content doubles to 4N EdU Uptake Occurs here.
G2: Period of cell growth where the cells are maintained at 4N
M: Cell division resulting in two daughter cells each with 2N nuclear DNA content
G2
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Click-iT™ EdU cell proliferation : Flow Cytometry
7-AAD fluorescence
New azide
labeled with
Alexa Fluor® 488 dye
Traditional 7-AAD
cell cycle dye
Hela cells:
Human cervical carcinoma cells
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Measure S phase entry with Click-iT™ EdU. HCS duplicates Flow data!
• Images: HeLa cells, EdU labeling for 120 min.; formaldehyde fixed and processed for Click-iTTMEdU reaction; DNA -Hoechst 33342; EdU –Click-iTTM Alexa Fluor®488 Azide Dye.
• Histograms show relative frequencies; “EdU positive” is green (bottom right); dynamically linked graph of DNA profile (bottom left) shows the distribution of “EdU positive” in DNA profile.
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Click-iT™ EdU Kits
A102082 plate (~200 tests)
Click-iT™ EdU Alexa Fluor® 647 High-throughput Imaging (HCS) Assay Kit
2 plate
(~200 tests)
2 plate
(~200 tests)
50
50
50
Number Tests
A10202Click-iT™ EdU Alexa Fluor® 647 Flow Cytometry Assay
A10034Click-iT™ EdU Pacific Blue™ Flow Cytometry Assay
A10044EdU (5-ethynyl-2’-deoxyuridine)
A10209Click-iT™ EdU Alexa Fluor® 594 High-throughput Imaging (HCS) Assay Kit
A10027Click-iT™ EdU Alexa Fluor® 488 High-throughput Imaging (HCS) Assay Kit
C35002Click-iT™ EdU Alexa Fluor® 488 Flow Cytometry Assay
Catalog NumberProduct
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Countess™ automated cell counter
NEVER USE A HEMOCYTOMETER AGAIN!
Accurately counts cells in 30 seconds
Calculates % viability
Measures average cell size
Includes dilution calculator
Call for a demo today
Automated cell counting at your fingertips
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Countess is a complete counting platform
C10227 Countess automated cell counter)– Includes Countess instrument, box of 50 slides and trypan blue
C10228 Countess™ cell counting chamber slides, 50– Includes trypan blue
*C10288 Countess™ field test kit (for internal sales only)– Includes box of 50 slides, trypan blue, beads and instructions
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Countess™ makes cell counting easy
3. Focus and Press “Count cells”
2. Insert slide into Countess instrument
1. Mix 10 uL of sample with 10 uL of trypan blue & pipet into Countess chamber slide
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Countess data has a wider range than hemocytometer
1.00E+05
1.00E+06
1.00E+07
1.00E+05 1.00E+06 1.00E+07
Cell suspension dilution
Mea
sure
d co
ncen
tratio
n
Countess™ automatedcell counterHemocytometer
Countess Range1x104 – 1x107 cells/mL
– Highest accuracy: – 1x105 – 4x106 cells/mL
Hemocytometer range– 2.5x105 – 2.5 x106
cells/mL
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The Countess works with many cell types
Cell type Species Tissue Primary or Immortalized Cell line Average cell size
293-Herg Human kidney immortalized 13 umA431 Human skin immortalized 15.5 umCHO-M1 Hamster ovary immortalizedCHSE Fish embryonic 16-17 umCOLO-207 Human intestine immortalizedCOS-7 Monkey kidney immortalizedHASMC human smooth muscle primary 20 umHeLa Human cervix immortalizedHepG2 Human liver immortalized 18 umHL-60 Human blood immortalizedHPAEC human artery endothelial primary 13 umHPASMC Human smooth muscle primary 20 umHUVEC Human umbilical vein primary 17 umJ774(MMM) Mouse blood immortalized 13-14 umJurkat Human blood immortalized 12 umK-562 Human bone/marrow immortalizedMCF-7 Human mammary immortalized 20-24 umMRC-5 Human lung immortalized 18 umNIH/3T3 Mouse embryonic immortalized 18 umPC-12 Rat adrenal immortalized 12-14 umSF-21 insect ovaryU266 Human blood immortalized 12-13 umU2OS. Human bone/marrow immortalizedAdipocytes human fat primary 16-17 ummelanocytes Human skin primarykeratinocytes Human skin primary
Counts cell sizes 5 – 60 microns
Concentration range is 1 x 105 – 4 x 106
cells/mL
Does it count clumpy cells?
Yes, it can count clumps of 2-5 cells, but
not larger clumps
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The Countess makes your science better
BETTER ACCURACY Minimize subjective counting. Can easily count more cells to increase accuracy
BETTER DOWNSTREAM RESULTS You are more likely to count when you “should,”rather than skip this step
INCREASE YOUR VERSATILITY Broadens capabilities for experimental design –easy to count many samples
FASTER COUNTING Takes 30 minutes to count and archive 30 samples
BIOHAZARD ISSUES Disposable slides better for biohazardous materials
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