Flow Cytometry Workshop

Insert Date

Dr Gareth Howell
g.j.howell@leeds.ac.uk
x37270
Workshop: Flow Cytometry

LBFF: Leeds Bioimaging

and Flow Cytometry Facility
Workshop – Flow Cytometry:
Basic concepts, applications and
experimental design
Workshop: Flow Cytometry

LBFF: Flow Cytometry Facility Details

Location: Garstang level 8

Manager: Dr Gareth Howell
http://www.fbs.leeds.ac.uk/facilities/
flowcytometry/
E: g.j.howell@leeds.ac.uk
T: x37270
My Office
Workshop: Flow Cytometry

BD FACSAria
BD FACSCalibur 2-laser, 7 colour analyser and cells sorting cytometer
2-laser, 4 colour analyser cytometer Interchangable emission filter set-up
Fixed emission filter set-up

Partec PASIII
Single laser, 4 colour analyser cytometer
HBO (mercury) lamp
Interchangable filter set-up
Workshop: Flow Cytometry

Purpose of this workshop:

To introduce the concepts of flow cytometry (FACS)analysis
To illustrate the role flow can play in your research
Demonstrate the capabilities of flow
Experimental design
To discuss the limitations of flow

Seminar:
Introduction to flow
Applications available

Practical demonstration:
flow applications and cell sorting
Workshop: Flow Cytometry

• What is flow cytometry (FACS or FCM)?

• Components
• Light scatter parameters
• Fluorescence and Multicolour
• Cell cycle analysis
• Apoptosis and necrosis assay
• Cell proliferation assay
• Sorting
Workshop: Flow Cytometry

• 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
Workshop: Flow Cytometry
Workshop: Flow Cytometry

Components of a flow cytometer Electronics

Fluidics

Optics
(detectors)
Optics
(lasers)
Workshop: Flow Cytometry

FLUIDICS
• 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
Workshop: Flow Cytometry

Components of a flow cytometer Electronics

Workshop: Flow Cytometry

Voltage
Low signal height
Laser
High signal height
Time

Laser Voltage

Time

Count
Voltage

h
Laser

Time
Intensity
Workshop: Flow Cytometry

Size and granularity using flow cytometry

Side
scatter

Forward
scatter
Workshop: Flow Cytometry

Cytometer Optical system comprises:

Dichroics and Filters

Fluidics

Detectors
Workshop: Flow Cytometry

Fluorescence
Emitted fluorescence intensity is proportional to binding sites

FITC FITC
FITC

FITC
FITC
FITC FITC
FITC
Number of Events

FITC
FITC

Log scale of Fluorescent Intensity

Workshop: Flow Cytometry

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.
Workshop: Flow Cytometry

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

* tandem dyes
Workshop: Flow Cytometry

Compensation FITC-Fluorescence Overlap

FITC PE PerCP
530/30 585/42 670/LP

PE
Relative Intensity

FITC

PerCP
500nm 550nm 600nm 650nm 700nm

Wavelength (nm) FITC

Workshop: Flow Cytometry

Perform
Compensation

FITC FITC

FITC PE PerCP
530/30 585/42 670/LP
Relative Intensity

24.8% of the FITC signal subtracted from PE.

On a FacsCalibur flow cytometer, there is no
provision to subtract FITC signal from PerCP.

500nm 550nm 600nm 650nm 700nm

Wavelength (nm)
Workshop: Flow Cytometry

Compensation PE-Fluorescence Overlap

FITC PE PerCP
530/30 585/42 670/LP

PE
Relative Intensity

FITC

PerCP
500nm 550nm 600nm 650nm 700nm 750nm 800nm

Wavelength (nm) PE
Workshop: Flow Cytometry

Optimal Under Over

Compensation Compensation Compensation

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

Workshop: Flow Cytometry

Applying Gates for sub-population analysis

Simple gating stratagies…

Gate on lymphocytes Assess T-cell population

Whole blood light scatter (fluorescence)
(light scatter)
…to more
complex!
Workshop: Flow Cytometry

Applications of flow cytometry in research

• Multicolour analysis
• Cell cycle
• Cell proliferation
• Apoptosis and Cell Viability
• Cell Sorting
• Multiplex analysis
Workshop: Flow Cytometry

Applications of flow cytometry in research

• Multicolour analysis
• Immunophenotyping
• Cells surface antigen detection (e.g. receptors, adhesion
molecules)
• Intracellular staining
• Assessing infection/transfection levels
• Antibodies/ dyes/ Quantum dots
Workshop: Flow Cytometry

• Immunophenotyping
e.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
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
Workshop: Flow Cytometry

•Cell Cycle Analysis

DNA probes The cell cycle

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 Note the cell volume (size) and DNA
concentration change as the cell progresses
through the cell cycle
Workshop: Flow Cytometry

Stoichiometric DNA probe binding

A typical DNA histogram

l
Workshop: Flow Cytometry

H x W = Area
W

Time

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
Workshop: Flow Cytometry

Cell Cycle Analysis:

Bromodeoxyuridine (BrdU) incorporation

•A limitation to standard single colour DNA

BrdU-FITC
S-phase
staining is that we can’t determine whether S-
phase cells are actually cycling

G1 G2

•Cells take up BrdU during S-phase, but not

during G1 or G2, an Ab vs BrdU then allows PI

us to determine which cells are actively

cycling within a population by two-colour
analysis:

hLimitations. hInvitrogen ‘Click-it’ EdU system

Workshop: Flow Cytometry

Assessing cell proliferation: BrdU incorporation

Pulse-label with BrdU and

taking samples at specific time
points allows us to determine
how cells behave kinetically
through the cell cycle.
Workshop: Flow Cytometry

Assessing cell proliferation using flow cytometry

CFSE loaded cells
Apoptosis and Cell Viability
Workshop: Flow Cytometry

•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)
Workshop: Flow Cytometry

Sub G1 apoptosis assay

DNA fragmentation allows apoptosis to

be quickly assessed with eg. PI
Can be seen as a population of small Sub-G1 peak
peaks to the left of G1 in a histogram
Quick and easy way to determine if
apoptosis is occurring
Workshop: Flow Cytometry

Apoptosis detection using viability dye uptake

Changes in membrane permeability due
to apoptosis allow intracellular dyes to
stain unfixed cells

7-AAD (DNA)

Live cells exclude dye

Apoptotic cells stain 7-AADdim

Dead cells stain 7-AADbright

Workshop: Flow Cytometry

Annexin-V/PI assay for apoptosis:

hPS normally on inside of cellular membrane hAnnV can bind to externalised PS
highlighting cells that are apoptotic hPI will only go into cells with compromised
membranes – dead (necrotic) cells

AnnV-FITC

PS

PI
Workshop: Flow Cytometry

•Apoptosis – Organelle Analysis

•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 much used pairing of annexin V and propidium
iodide
Workshop: Flow Cytometry

•Mitotracker Red can be loaded into live cells and taken up by mitochondria
•Loss of membrane potential causes apoptotic cells to loose dye from organelle
•Shift in fluorescence intensity indicates compromised mitochondria

(CCCP) carbonyl cyanide m-chlorophenyl hydrazone

Alternative: DiOC6(3) for green fluorescent labelled mitochondria

Workshop: Flow Cytometry

Yeast cells + TOPRO-3

Live/Dead assay
Utilise the properties of dyes that are
impermeable to intact cell
membranes:
Propidium iodide
DAPI
TOPRO-3

+ve fluorescence indicates

compromised cell membranes and
therefore dead cells
Live cells retain their
morphology and
Dead cells show more
appear larger in size
granularity and reduced
and less granular
size
• 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
Cell Sorting
Workshop: Flow Cytometry

• 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%
Workshop: Flow Cytometry

Cell Sorting Transfected Cells

• Improve transefection efficiency • Single cell cloning

• siRNA knock down • Isolate spcific cell types from tissue preps
• Stable cell line production • Up to 4 populations simultaneously
• Rare population isolation • Various collection tubes and plates
Workshop: Flow Cytometry

Multiplex beads
Workshop: Flow Cytometry

Fluorescent capture bead technology

• Beads of various fluorescent intensities
• Can be conjugated with antibodies or biotin
• Multiplex conjugated kits

Bender MedSysytems
Beckton Dickinson
Beckman Coulter
Luminex
Qiagen
Upstate

• ELISA principals
Workshop: Flow Cytometry

Coated latex bead

(FL1)
Y

FL1
Y

Incubate with e.g. cell lysate

FL2

Analyse by flow
cytometry using
Y

bivariant dot plot

Incubate with FL2
labelled antibody vs Y
protein of interest
Workshop: Flow Cytometry

Fluorescent proteins and their applications in bioimaging

Workshop: Flow Cytometry

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)
Workshop: Flow Cytometry

Disadvantages of fluorescent
proteins?
•Size •Always ensure adequate controls
•Artefacts •N and C terminus constructs
•Check functionality vs WT (if possible)
•Mis-targetting
•Don’t always select/gate brightest cells! Be
•Over expression objective
•Cell toxicity •Stable cell lines? Transgenics?
•Alternative expression vector
•pH sensitive
Workshop: Flow Cytometry

• 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.