Introduction into Flow Cytometry

Introduction into

Flow Cytometry

Stephanie Gurka, Andreas Hutloff, Timo Lischke,

• Use of fluorescence marked monoclonal antibodies

multi-parameter analysis (up to 18) for each individual cell

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

• Blood cells

• Tissue cells

• Algae

• Protozoa

• Chromosomes

• Yeast

Prerequisite:

single cell suspension

(Disaggregation: mechanical or enzymatic)

Instrument Overview

Sample

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

Multi-Laser Systems

488 nm Laser

405 nm Laser

633 nm Laser

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

v ol ta ge time v ol ta ge v ol ta ge

10 9 8 7 6 5 4 3 2 1 0 C e ll c o u n t

is most influenced by the size of cells

FSC-Histogram

FSC-Threshold

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

• 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

• 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 cells Detection 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 nm

FITC

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

Properties of Fluorochromes

Laser

SSC detector

photo multiplier tube (PMT)

FCS detector

photo diode

signal levels are high

Fluorescence detection

Fluorescence detector

(PMT3, PMT4 etc.)

FCS detector

photo 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

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

Stain Index = D / W

Resolution Sensitivity:

W2

W1

D

Reagent Filter Stain Index

PE 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)

Histogram:

Data Analyis

2D plot:

Intensities of 2 Light/Fluorescence Parameters plotted against each other

C

D

Gating - Statistics

MFI =10

MFI =150

Quadrant Statistic

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.

Fluorescence dyes used for flow cytometry

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

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

Fluorescence dyes used for flow cytometry

Fluorochrome

Company

FITC, PE, PerCP, APC, Cy5

Becton Dickinson

Alexa Fluor ___

Molecular Probes

(Invitrogen)

eFluor ___

eBioscience

V ___

BD Horizon

Pacific ___

___

Oregon ___

___

…

…

Fluorescence: points to consider

pH dependence

Quenching

Photobleaching

FITC

Photostability / Photobleaching

Brightness (high quantum yield)

Photostability (no bleaching)

pH insensitivity (stability of fluorescence emission)

Water solubility (little hydrophobic interactions)

Instrument compatibility (fit to excitation wavelenght)

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.

I)

g

I)

Large Protein dyes:

phycoerythrin, allophycocyanin, peridinin-chlorophyll-protein

Coupling of fluorescent dyes to antibodies

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

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

•

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

Antibody titration basics

1 10 100 10001 10 100 1000 10000 _signal noise S:N ng antibody Intensity i n c re a si n g a m o u n t o f m A b

Basis of multicolor flow cytometry

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.

Two Color Experiment - 1 Laser

Filters collect 2 colors

positive population negative population

Fluorescence Compensation

mathematical subtraction

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

Small errors

in compensation of a dim control can result

in 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 Ab

to 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 One

leaving 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.

based on fluorescence height, fluorescence area and signal width.

Autofluorescence

• fluorescent signals generated by the cells themselves

• Present in all cells (viable and dead).

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

• observed in all fluorescence channels,

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

Signal Separation: different fluorochromes

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.

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/

staining procedure:

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.

- 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 populations

into 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.

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)

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

sensitivity and throughput rates enable detection of extremely rare populations and

events (frequencies < 10

_),

■ Hematopoietic stem cells ■ Dendritic cells

■ Residual disease detection (tumor cell enumeration) ■ Antigen-specific T cells

■ Transient transfectants

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,