Download - Workshop: Flow Cytometry LBFF: Leeds Bioimaging and Flow Cytometry Facility Workshop – Flow Cytometry: Basic concepts, applications and experimental design.

Workshop: Flow Cytometry

LBFF: Leeds Bioimaging and Flow Cytometry Facility

Workshop – Flow Cytometry:

Basic concepts, applications and experimental design

LBFF: Flow Cytometry Facility Details


Location: Garstang level 8

Manager: Dr Gareth Howellhttp://www.fbs.leeds.ac.uk/facilities/flowcytometry/

E: [email protected]

T: x37270My Office


BD FACSCalibur2-laser, 4 colour analyser cytometerFixed emission filter set-up

BD FACSAria2-laser, 7 colour analyser and cells sorting cytometerInterchangable emission filter set-up

Partec PASIIISingle laser, 4 colour analyser cytometerHBO (mercury) lampInterchangable filter set-up

Purpose of this workshop:

To introduce the concepts of flow cytometry (FACS)analysisTo illustrate the role FACS can play in your research

Demonstrate the capabilities of FACSExperimental design

To discuss the limitations of FACS

Seminar:Introduction to FACSApplications available

Practical demonstration:FACS applications and cell sorting


• What is flow cytometry?• Components• Size and complexity using flow cytometry• Fluorescence and Multicolour flow cytometry • Cell cycle analysis• Apoptosis and necrosis assay • Cell proliferation assay• Sorting


• What is flow cytometry?– The analysis of single particles, often cells,

within a heterogeneous suspension

• Whole blood, Cell cultures, Separated tissue, Isolated nuclei, Bacteria/yeast/parasites, Algae & plankton

• Signal from individual particles is collected for analysis as they pass through a laser in a stream of fluid.

• Data displayed as events on histograms/dot plots



http://upload.wikimedia.org/wikipedia/commons/c/c7/Picoplancton_cytometrie.jpg

Components of a flow cytometer

Fluidics

Electronics

Optics(detectors)

Optics(lasers)


• Vital that cells pass through the

laser bean in single suspension• Cells injected into a flowing

stream of saline solution (sheath

fluid)• Hydrodynamic focusing• Compresses cell stream to

approx 1 cell diameter• Allows single cells to be

interrogated by the laser•Optimal ‘imaging’ of cells is

achieved with a ‘low’ flow rate

and high concentration of

sample

FLUIDICS


Components of a flow cytometer Electronics


LaserLaser

LaserLaser

LaserLaser

Time

Volt

age

Time

Volt

age

Time

Volt

age

Intensity

Cou

nt

h

Low signal height

High signal height


Forward scatter

Side scatter

Size and complexity using flow cytometry


Cytometer Optical system comprises:

Fluidics

Dichroics and Filters

Detectors


FITC FITC

FITC

FITC

FITC

FITC

FITC

FITC

FITC

Emitted fluorescence intensity is proportional to binding sites

FITC

Log scale of Fluorescent Intensity

Num

ber

of E

vent

sFluorescence


FACS machines use lasers as sources for excitation; fixed single wavelength. Fluorescent light emission collected using filters as before. Therefore have to use flurophores compatible with lasers employed: FACSCalibur/FACSAria 488 and 647nm lasers.

APC


Emission is collected through emission filters positioned within the optical system of the flow cytometer.

APC


Dyes suitable for use on flow cytometers:

• 488 excitation:– FITC, Alexa 488, GFP, YFP– PE, PI, RFP, – PerCP, 7-AAD, PE-Cy5, PE-Cy7

• 633nm excitation:– APC, TOPRO-3, Cy5, Cy7

Compensation FITC-Fluorescence Overlap

FITC

PE

FITCP

erC

P

650nm 700nm500nm 600nm

Re

lati

ve

In

ten

sit

y

Wavelength (nm)

550nm

PerCP670/LP

FITC 530/30

PE585/42


FITC

PE

650nm 700nm500nm 600nm

Re

lati

ve

In

ten

sit

y

Wavelength (nm)

550nm

24.8% of the FITC signal subtracted from PE.

On a FacsCalibur flow cytometer, there is no provision to subtract FITC signal from PerCP, referred to as cross-beam compensation.

FITC

PEPerform

Compensation

PerCP670/LP

FITC 530/30

PE585/42


Compensation PE-Fluorescence Overlap

FITC

PE

650nm 700nm500nm 600nm

Re

lati

ve

In

ten

sit

y

Wavelength (nm)

550nm 750nm 800nm

PEP

erC

P

PerCP670/LP

FITC 530/30

PE585/42


Optimal

Compensation

Under

Compensation

Over

Compensation

16-colour compensation possible now on latest 3-laser, multi-parameter cytometers


Applying Gates for sub-population analysisSimple gating stratagies…


Whole blood light scatterGate on lymphocytes

(light scatter)Assess T-cell population

(fluorescence)

…to more complex!

Applications of flow cytometry in research

• Immunophenotyping• Stem cell characterisation• Cell cycle• Apoptosis and Cell Viability• Cell proliferation (CFSE, BrdU/Hoechst)• Cell Sorting


• Immunophenotypinge.g. diagnosis of leukaemia

COMBINATION POPULATION IDENTIFIED CD4+/CDw29+ Helper/effector, more mature memory cells CD4+/CD45R+ Suppressor inducer, less mature non-memory cells CD4+/Leu8+ Suppressor inducer, some helper function CD4+/Class II MHC Activated cells, immature cells CD4+/CD25+ Activated cells (IL2 receptor) CD4+CD38+ Immature cells, activated cells CD8+/CD11b+ Of the CD11b+ cells the suppressors are bright CD8+ and NK are dim CD8+ CD8+/CD28+ Cytotoxic precursor/effector cells CD8+/CD57+ Cytotoxic function CD8+/Class II MHC+ Activated cells, immature cells CD8+/CD25+ Activated cells (IL2 receptor) CD8+/CD38+ Immature cells, activated cells CD16+/CD57+ Low NK activity CD16+/CD56+ Most potent NK activity


Stem Cell Characterisation

• Stem Cell Characterisation Functional analysis

• Cytosolic aldehyde dehydrogenase (ALDH) activity• High levels found in stem cells• Drug resistance• Cleavable enzyme assay (AldeFluor, StemCell Tech.)

http://science.cancerresearchuk.org/

• Stem Cell Characterisation Side population analysis

• Efficient membrane pumps

• Exclude dyes e.g. Rhodamin 123 and Hoechst dye

• Hoechst dyes bind DNA in live cells (blue and red fluorescence)

• UV excitation

• Pumped out by ABC (ATPase Binding Cassette)

• Stem cells can be characterised by low side populations –ve for Hoechst dye.

• Membrane markers to confirm. http://science.cancerresearchuk.org/

• Stem Cell Characterisation Clinical Application – CD34+ Stem Cell Enumeration

• Method of repopulating stem cells following radiotherapy treatment

• Patient treated to produce excessive levels of pluripotent cells which are harvested from peripheral blood

• Number of cells reintroduced important in succsss rate of procedure

• Abs vs stem cell markers CD34 and CD45 used in enumeration procedure

Cell Cycle Analysis

•Cell Cycle Analysis

DNA probes

DAPI }Hoechst } UV

Propidium iodide (PI) }7-AAD } 488

TOPRO-3 }DRAQ5 } 633

These dyes are stoichiometric – number of bound molecules are equivalent to the number of DNA molecules present

The cell cycle

Note the cell volume (size) and DNA concentration change as the cell progresses

through the cell cycle


l

A typical DNA histogram

Stoichiometric DNA probe binding


Measuring height against width gives us area

Two G1 cells together will have the same PI intensity as a G2 cell, but the area (signal h x w) will be greater and therefore can be discriminated on a plot of signal width vs area

Time

Intensity H

H x W = AreaW


Cell Cycle Analysis:Bromodeoxyuridine (BrdU) incorporation

•A limitation to standard single colour DNA staining is that we can’t determine whether S-phase cells are actually cycling

•Cells take up BrdU during S-phase, but not during G1 or G2, an Ab vs BrdU then allows us to determine which cells are actively cycling within a population by two-colour analysis:

PI

Brd

U-F

ITC S-phase

G1 G2

Limitations. Invitrogen ‘Click-it’ EdU system


Pulse-label with BrdU and taking samples at specific time points allows us to determine how cells behave kinetically through the cell cycle.


Apoptosis and Cell Viability

•Apoptosis• Gene directed cell death

• An event that occurs during development and a response to trauma or disease

• Cancer cells develop a strategy to evade apoptosis

Apoptosis results in a number of cellular events that can be analysed by FACS:

•Fragmentation of DNA (subG1 assay, Hoechst dyes)

•Membrane structure and integrity Annexin-V, PI)

•Mitochondrial function (Mitotracker Red)

•Caspase activity (antibodies assay)


• Quick and easy apoptosis assay: Sub-G1

Sub-G1 peakDNA fragmentation allows apoptosis to be quickly assessed with eg. PICan be seen as a population of small peaks to the left of G1 in a histogramQuick and easy way to determine if apoptosis is occurring


Annexin-V/PI assay for apoptosis:PS normally on inside of cellular membrane AnnV can bind to externalised PS highlighting cells that are apoptotic PI will only go into cells with compromised membranes – dead (necrotic) cells

AnnV-FITC

PS

XXX

XX

X

PI


•Membrane potential of the organelle reduced•Mitochondrial activity appears to change in parallel with cytoplasmic and plasma membrane events•Dyes that accumulate in mitochondria can therefore play role in detecting apoptosis

-Mitotracker Red CMXRos-JC-1-DiOC2(3)-Laser Dye Styryl-751 (LDS-751)

•Reagent combinations can provide a window on intracellular processes not available with the muchused pairing of annexin V and propidium iodide

•Apoptosis – Organelle Analysis


(CCCP) carbonyl cyanide m-chlorophenyl hydrazone

•Mitotracker Red can be loaded into live cells and taken up by mitochondria

•Loss of membrane potential causes apoptoic cells to loose dye from organelle

•Shift in fluorescence intensity indicates compromised mitochondria


Alternative: DiOC6(3) for green fluorescent labelled mitochondria

Live/Dead assay

Utilise the properties of dyes that are impermeable to intact cell membranes:

Propidium iodideDAPITOPRO-3

+ve fluorescence indicates compromised cell membranes and therefore dead cells

Yeast cells + TOPRO-3

Dead cells show more granularity and reduced size

Live cells retain their morphology and appear larger in size and less granular


• Cell mediated cytotoxicity assay

• Dye exclusion assay to assess cell death, PKH26 (Sigma)

• Example: tumour cells (target) and NK cells (effector)

• Positive cytotoxic event recorded as an increase in cell fluorescence

• No requirement for radioisotopes e.g. 51Cr-release assay

• Also cell by cell assay - accurate Single parameter histograms

Assessing cell proliferation

Assessing cell proliferation using flow cytometry

CFSE loaded cells


Assessing cell proliferation using flow cytometry

BrdU/Hoechst quenching assay

DNA binding dye Hoechst fluorescence quenched if BrdU incorporated into DNA

Can be used to assess cell proliferation

PI not quenched – allows determination of cell cycle as before.

Requires flow cytometer with UV excitation

Needs careful optimization of BrdU labellingDiermeier et al (2004) Cell Prolif. 37:195

• Cell sorting– Allows rare populations to

be isolated from heterogenous populations (cell culture, blood samples, etc)

– Can isolate sub cellular particles (e.g. endosomes, nucleus, chromosomes)

– Allows transfection experiments to be enriched and single cell clones to be isolated

– Can produce purity >95%


Cell Sorting Chromosomes

• Chromosome specific DNA libraries, DNA for sequencing, probes for reverse painting, array painting.

• Many lymphomas have chromosomal abnormalities.

• Base specific dyes allow chromosomes to be separated on dot plots

http://www.chrombios.com/Service/ServiceFACS.html

Fluorescent proteins and their applications in bioimaging


What can we do with fluorescent proteins?

•Use as reporter genes to identify gene activation

•Study transfection rates / success

•Expression of tagged proteins

-Placed in-frame with gene of interest

•Compare expression / localisation against function (combine FACS with

imaging)

•Environmental indicators (pH)

•Protein-protein interactions (FRET, split-GFP)


Disadvantages of fluorescent proteins?

•Size

•Artefacts

•Mis-targetting

•Over expression

•Cell toxicity

•pH sensitive

•Always ensure adequate controls

•N and C terminus constructs

•Check functionality vs WT (if possible)

•Don’t always select/gate brightest cells! Be

objective

•Stable cell lines? Transgenics?

•Alternative expression vector


• Summary– Flow cytometry is a powerful method for rapidly

quantitating cellular fluorescence– A number of functional assays such as cell cycle and

apoptosis can be determined by flow and can be used as a method for assessing e.g. the effects of drugs on cell function, or the expression of mutant proteins

– Finally, cells and sub-cellular particles can be sorted from heterogeneous samples to yield near homogeneous populations for subsequent culturing or analysis.


Wavelengths of visible light

The wavelength of visible light ranges from 380 nm (violet) to 780 nm (red).

UV IRVisible light spectrum


Fluorescence - basics

Wavelength (nm)

Fluore

scence

inte

nsi

ty

Fluorescent molecules are characterised by their ability to absorb short wavelength light and emit at a longer wavelength.

Excitation Emission


Bandpass eg 530/30 FITC, Alexa 488

Bandpass eg 585/40 Phycoerythrin (PE)

Longpass eg LP670 Cy5, APC, PerCP

UV IRVisible light spectrum


Emission SpectraAPC PerCP

Wavelength (nm)400 500 600 700

100%

0%

Nor

mal

ized

Int

ensi

ty

FITC PE

800


More Emission Spectra!

APC PerCPPI

Wavelength (nm)400 500 600 700

100%

0%

Cascade Blue

Nor

mal

ized

Int

ensi

ty

FITC PEAlexa 430 PerCP-Cy5.5

800

PE-Cy7


Designing Multicolour Experiments Why?

•Allows a number of different structures (proteins / lipids / compartments) to be visualized at the same time

• Can provide clues / evidence to the function of your protein of interest

• Design principles can be applied to any fluorescent molecule: fluorescent protein, membrane marker, antibody or dye in live or fixed cells

How?

• Simply by studying the configuration of the imaging system and the excitation / emission characteristics of the proposed dyes one can design a multicolour fluorescent experiment


Graphic representations of fluorescence data

Green

Red

Green +ve/Red -ve

Green -ve/Red -ve

Green +ve/Red +ve

Green -ve/Red +ve


http://www.retrovirology.com/content/4/1/18/figure/F4?highres=y