Introduction into Flow Cytometry

Stephanie Gurka, Andreas Hutloff, Timo Lischke,

Kroczek-Lab, Robert Koch Institute, Berlin

• Use of fluorescence marked monoclonal antibodiesmulti-parameter analysis (up to 18) for each individual cell

• High flow-rate (> 20,000 cells/sec)

Flow Cytometry - FCM

FACS = Fluorescence Activated Cell Sorting(also used for analytical cytometers)

• Analysis of the physical properties of single cells or other biological particles

• Basic principle: a single cell passes through a flow cell and is illuminated by a laser source

detection and analysis of scattered / emitted light

• Blood cells• Tissue cells• Algae• Protozoa• Chromosomes• Yeast

Prerequisite: single cell suspension

(Disaggregation: mechanical or enzymatic)

Particles can be measured with a flow cytometer

Instrument Overview

Sample(single cell suspension)

The Laser System

typically used monochromatic laser sources (nm)

• Gas laser systems which require complex air or water cooling are more and more substituted with diode and solid-state lasers

• Modern Flow-cytometers (e.g. BD LSR II) accomodate up to five lasers

Multi-Laser Systems

488 nm Laser

405 nm Laser

633 nm Laser

17 ms 55 m

s

time delay

Coherent lightsource(488 nm)

Optics - Forward Scatter Channel (FSC)

detect the amount of light scattered in the forward direction

(along the same axis that the laser light is traveling)

Forward Scatter Detector

particle passes through the focus -> scattered light is detected by a photodetector,

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-> an electrical pulse is generated and presented to the signal processing electronics.

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FSC-Histogram

Intensity of forward scatter

FSC-Threshold

• instrument is triggered when the signal exceeds a predefined threshold level

-> reject non-particle events such as debris/noise from optical / electronic sources.

FSC tends to be more sensitive to surface properties of particles (e.g. cell ruffling)-> can be used to distinguish live from dead cells

• Intensity of SSC is most influenced by the shape and optical homogeneity of cells

Optics - Side Scatter Channel (SSC)

• detect the amount of light scattered to the side (90° to the axis that the laser light is traveling)

Scatter Plot

SSC tends to be more sensitive to inclusions within cells-> can be used to distinguish granulated cells from non-granulated cells

• the relative size (FSC)

• the relative granularity

or complexity (SSC)

• the fluorescence intensity (FL1/2,

up to

FL X)

->

Which parameters can be measured?

Analysis of complex primary samples (heterogeneous cells), such as immune cellsDetection of rare cell types

Using Fluorescence in Flow Cytometry

or cells transfected with fluorescent proteins

• Nucleic acid fluorochromes• Fluorochromes for membrane potential analysis or for ion flux (e.g. Ca2+)• Membrane label fluorochromes

What is Fluorescent Light ?

488 nmFITC

530 nm

Incident Light Emitted Fluorescent Light

The fluorochrome absobs energy from the laser and releases the absorbed energy by:

a) Vibration and heat dissipation

b) Emission of photons of a longer wavelength =====> FLUORESCENCE

Stokes shift: energy difference between the wavelength of absorption and emission

Stokes Shift

Properties of Fluorochromes

Excitation/Emission Spectra

Laser

SSC detector photo multiplier tube (PMT)

FCS detectorphoto diode

signal levels are high

Fluorescence detection

Fluorescence detector(PMT3, PMT4 etc.)

FCS detectorphoto diode

signal levels are high

SSC detector photo multiplier tube (PMT)

• Specificity controlled by the wavelength selectivity of optical filters and mirrors.

• Fluorescence emitted by each fluorochrome is usually detected in a unique fluorescence channel.

Optical Layout - BD FACSCalibur

D = difference between positive and negative peak mediansW = 2 x rSD (robust standard deviation)

Stain Index = D / W

Resolution Sensitivity:

W2

W1

D

Reagent Filter Stain IndexPE 585/40 356.3

Alexa 647 660/20 313.1

APC 660/20 279.2

PE-Cy7 780/60 278.5

PE-Cy5 695/40 222.1

PE-Alexa 610 610/20 80.4

Alexa 488 530/30 75.4

FITC 530/30 68.9

APC-Cy7 7801/60 42.2

Alexa 700 720/45 39.9

Pacific Blue 440/40 22.5

Clone RPA-T4

Fluorescence One Color Histogram

in conjunction with fluorescence-based protein reporters (GFP)-> monitor both transfection efficiency and protein expression levels.

Histogram:

Data Analyis

Light/Fluorescence Intensity

cell

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2D plot:

CD4

Intensities of 2 Light/Fluorescence Parameters plotted against each otherC

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Gating - Statistics

X

Y

% Y+X- % Y+X+

% Y-X-% Y-X+

% A

% B% C

MFI =10 MFI =150

Quadrant Statistic

Mean Fluorescence Intensity (MFI)

Gating - example

Gating - example

A laser beam of a single wavelength is used to excite several fluorochromes with different Stokes shifts and, thereby, produce a variety of fluorescent colors.

Basis of multicolor flow cytometry

Fluorescence dyes used for flow cytometry

http://www.bdbiosciences.com/spectra/

Fluorochrome excitationwavelength (nm)

emission maximum (nm)

LP Mirror (Canto2)

BP Filter (Canto2)

FITC, CFSE 488 525 502 530/30

PE 488 575 556 585/42

PI 488 620

PerCP / PerCP-Cy5.5 / PE-Cy5.5 488 675 / 695 / 695 655 670LP

PE‑Cy7 488 767 735 780/60

AF647 / Cy5 / APC 633 665 / 667 / 660 685 660/20

A700 633 723 710 730/45

APC-Cy7 633 767 735 780/60

Pacific Blue / DAPI 405 451 / 460 - 450/50

Pacific Orange / DAPI 405 551 / 460 502 510/50

Knowing the excitation and emission properties of fluorescent compounds: Select combinations of fluorochromes that will work together optimally on a specific flow cytometer with specific lasers !

Fluorescence dyes used for flow cytometry

Fluorochrome CompanyFITC, PE, PerCP, APC, Cy5 Becton Dickinson

Alexa Fluor ___ Molecular Probes (Invitrogen)

eFluor ___ eBioscienceV ___ BD Horizon

Pacific ___ ___Oregon ___ ___

… …

Fluorescence: points to consider

pH

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Concentration fluoresceinFl

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Time (sec)

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Photostability / Photobleaching

Molecular Probes

Brightness (high quantum yield)

Photostability (no bleaching)

pH insensitivity (stability of fluorescence emission)

Water solubility (little hydrophobic interactions)

Instrument compatibility (fit to excitation wavelenght)

Multiparameter (small emission spectrum)

Wanted properties of fluorochromes

Types of Fluorochromes

I) Small dyes:FITC, Cy5, AlexaFluor´s, eFluor´s,

accessory photosynthetic pigment of red (R-PE, PerCP) or bluegreen algae (APC).

PE: 240-kDa protein with 34 phycoerythrobilin fluorochromes per molecule.APC: 105-kDa protein with 6 phycocyanobilin chromophores per molecule.PerCP: 35-kDa protein with phycoerythrobilin fluorochromes

I) gI) Large Protein dyes:

phycoerythrin, allophycocyanin, peridinin-chlorophyll-protein

FITC

PEB

Coupling of fluorescent dyes to antibodies

I) Small dyes / haptens (FITC, Cy5, AFs, Dig, …)

Reaction with primary amine groups of the mAb

fluorescein-5-EX succinimidyl ester

II) Protein dyes (phycoerythrin, allophycocyanin)

Amine - thiol crosslinking

1) The bifunctional crosslinker Succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC) reacts with amine groups of the fluorescent protein (R1)thereby introducing a maleimide group

2) The mAb (R2) is partially reduced (with DTT) which yields free sulfhydryl groups

Coupling of fluorescent dyes to antibodies

• maximize signal:noise (pos/neg separation)– This may occur at less than saturated staining– This may or may not be the manufacturer’s recommended titer

• Titer is affected by:– Staining volume – Number of cells– Staining time and temperature– Type of sample (whole blood, PBMC, etc.)

Antibody titration basics

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Fluorescence Tandem

Basis of multicolor flow cytometryA laser beam of a single wavelength is used to excite several fluorochromes with different Stokes shifts and, thereby, produce a variety of fluorescent colors.

Two Color Experiment - 1 Laser

Filters collect 2 colors

positive population negative population

positive population negative population

Fluorescence Compensation

mathematical subtraction of the fluorescence due to one fluorochrome from the fluorescence due to another

PE-MFI (neg) = PE-MFI (pos)

Fluorescence Compensation

Fluorescence Compensation

Small errorsin compensation of a dim control can resultin large compensation errors with bright reagents

• Cells stained with a single fluorochrome-conjugated Ab (analyzed individually)

-> One control for each of the fluorochromes used in the experiment

+ Single control for every tandem conjugate

• Negative and positive populations are required (>10%)

• Use bright markers to setup proper compensation

Compensation controls

• manually (up to 4 FL) or automatic compensation (>4 FL)

• CompBeads

Specificity Controls

unstained / control: to detect "auto-fluorescence" or background staining (monocytes/macrophages, cultured cells, or activated cells)(to set up PMT-voltage for FSC, SSC and FL-channels)

secondary control: for indirect staining (Bio/SAv, Dig/anti-Dig) - secondary Ab alone to control for non-specific binding of this polyclonal Abto dead or sticky cells.

specificity (experimental and gating) controls:e.g. Transfected cells: transfected / mock transfected / wt cell line,

Primary cells: WT / KO or activated / naive

Controls must undergo the same treatment (i.e., preparation, fixation)as all the tubes in an experiment.

not necessary for (lineage) markers with clearly separated populations

Isotype Control: Ab with the same Ig isotype as the test Ab, specificity known to be irrelevant to the analyzed sample

-> whether observed fluorescence is NOT due to non-specific (Fc receptors, dead cells) binding of the fluorescent Ab.

(one for each class of antibody used for staining, with the same concentration and F/P ratio as Ab of interest)

FMO Control: Fluorescence Minus Oneleaving out the antibody of interest in the staining panel

-> fluorescence spillover of all other fluorochromes in channel of interest.

„Cold Block“: Preincubation with an excess of unlabeled mAb prior to addition of fluorophore labeled mAb (no wash between)

All events (cells) with fluorescence above the threshold set with the above controls are considered positive for the marker of interest.

Further Specificity Controls

Comparison of gating controls

based on fluorescence height, fluorescence area and signal width.

Doublet discrimiation

Autofluorescence• fluorescent signals generated by the cells themselves (from pyridine and flavin nucleotides)

• Present in all cells (viable and dead).

• Adds to fluorescence label of cells -> decreases fluorescence detection limit

• observed in all fluorescence channels, but decreases dramatically at longer wavelengths (>600 nm, far-red/infra-red).

-> for cell types with high autofluorescence, a dye with a longer emission wavelength (APC, APC-Cy7) often provides excellent signal-to-noise ratio.

General principle: Dye reacts with free amines. Live cells (left) react with the fluorescent reactive dye only on their surface (weakly fluorescent cells). Cells with compromised membranes (dead, right) react with the dye throughout their volume (brightly stained cells).In both cases, the excess reactive dye is washed away.

Dead cell exclusion

Dead cells, with compromised membrane integrity, tend to be sticky-> bind all sorts of reagents unspecifically. -> exclude dead cells from analysis

• dye exclusion methods with DNA intercalating fluorochromes: propidium iodide (PI), 7-amino-actinomycin D (7-AAD) or

DAPI staining to positively identify dead cells by their membrane permeability

• fixable live/dead stain with fluorescent dye

Signal Separation: different fluorochromes

Isotype Control

FITC

PE

PE-Cy7

APC-Cy7

13.68

28.20

26.84

75.29

a- hu CD4 conjugates

important for multicolor analysis: choice of which antibody to use with which fluorochrome (often many "correct" combinations possible)

consider: For any given mAb clone, the signal-to-noise ratio (positive/negative) can differ depending on the fluorochrome and instrument used

• Blocking of Fc receptors with polyclonal Ig or specific mAb against Fc-Receptors (species specific!)

-> significantly reduces background staining, (usually not necessary with cell lines)

caution with indirect staining protocols and anti-rat-Ig (use purified mouse-gamma globulin or mouse serum instead)

Specificity / Non-Specificity: Fc-Receptors

Ab bind to many cell types by their non-specific (Fc) ends. Monocytes, BC and DC, professionally bind many Ab through their Fc-receptors.

• Fab or F(ab’)2 fragments

Sample preparation time, temperature, buffer (pH, salt concentration)Lysis, digestion, fixation, permeabilisation, washing steps

instrument number and type of Lasers, Filters, Fluorescence Detectors

-> fluorochromes/ -combinations

antibody clone, affinity, monoclonal vs. polyclonal, Ig-Isotype, type of Fluorochrome, concentration, F/P ratio, (may differ from lot to lot)

Cell number and concentration: depending on the number of events to be analyzed (due to cell loss during staining approx. 2 times more cells for staining than for analysis)

Cell concentration during staining: Maximum density for staining is 5x107 cells/ml -> 50 μl staining volume for up to 2.5 Mio cells

20- 30 min at 4°C in the presence of NaN3 to be sure of minimizing capping / internalization/

miscellaneous loss of surface-bound antibodies

staining procedure:

Rapid and scalable: Performed in 96-well plates and in parallel

Analytical Variables to consider

1) Choose brightest set of fluorochromes for particular instrument configuration.

1) Choose fluorochromes to minimize the potential for spectral overlap.

- high Compensation for adjacent channels, (FITC vs PE) - usually low Cross-beam compensation (blue vs red laser) Exceptions: GFP and very bright FITC signals like CFSE

(also excited by 405 nm detected in PacO channel); PE-Cy5.5 / PerCP-Cy5.5 (excited by 633 nm detected in AF700 channel)

3) Reserve the brightest fluorochromes for “dim” antibodies, and vice versa.

- Highly expressed Antigens will be resolved with almost any fluorophore - Antigens expressed at lower density might require brighter flurophores to separate the positive cells adequately from the unlabeled cells PacO < APC-Cy7 = PacB = FITC = AF700 = PerCP < PE-Cy7 < AF647 = PE = APC

1) Avoid spillover from bright cell populationsinto detectors requiring high sensitivity for those populations.

- Strongly expressed Antigens impair the sensitivity/signal resolution of the adjacent channel - Preferentially, use this channel for Antigens which are not on the same cell as the Ag of interest

5) Take steps to avoid tandem dye degradation, and consider its impact upon results.

Multicolor analysis: Choice of Ab-Fluorochrome

Set voltages: Decrease voltages for any detectors where events are off-scale

Increase voltages for any detectors where low-end resolution is poor

Analytical Variables to consider

Data Acquisition, Analysis and Interpretation

Instrument setup and performance • adjust and optimize PMT settings (optimal sensitivity)

Speed of analysis (high flow rate -> less intensity resolution)

• Run single-stained compensation controls for each experiment and set compensation• Run samples

• Run appropriate controls: Instrument setup controls (e.g., CompBeads)Gating controls (e.g., FMO)Biological controls (e.g., unstimulated samples, healthy donors)

Data Analysis / Interpretation appropriate number of acquired events to ensure reliable results gating strategy,

• Visually inspect compensation

Create a template containing dot plots of each color combination of the experiment, then examine a fully stained sample for possible compensation problems

• Check gating across all samples in the experiment. Gates may need to be adjusted across donors and/or experimental runs.

-> Avoid classification errors and false conclusions due to improper compensation and/or gating, or sample artifacts

Ask for interpreting the data, experiment and instrument setup

-> save time and labor

Analytical Variables to consider

sensitivity and throughput rates enable detection of extremely rare populations and events (frequencies < 10-6),

■ Hematopoietic stem cells■ Dendritic cells■ Residual disease detection (tumor cell enumeration)■ Antigen-specific T cells■ Transient transfectants

Rare Event Detection

Dump channels

use of an"dump channel" significantly improves detection of rare cells or resolution of dim stains (e.g. CD11c).

staining for an antigen not expressed by the cells of interest ("lineage negative„) -> exclusion of these cells for analysis

e.g. B220 for murine T cells,CD3 + CD8 + Ly-6G/C + CD11b for B cells, CD3 + CD19 for dendritic cells.

-> also exclude cells binding antibodies unspecifically.

Preferentially, the CasY or A700 channels are used as dump.