introduction into flow cytometry - bnitm · • basic principle: a single cell passes through a...
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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,
volta
ge
time
volta
gevo
ltage
-> an electrical pulse is generated and presented to the signal processing electronics.
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Intensity
Cel
l cou
nt
is most influenced by the size of cells
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
coun
t
2D plot:
CD4
Intensities of 2 Light/Fluorescence Parameters plotted against each otherC
D8
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
Rel
ativ
e in
tens
ity
Concentration fluoresceinFl
uore
senc
e in
tens
ity
Time (sec)
% in
ital f
luor
esen
ce in
tens
ity pH dependence Quenching PhotobleachingFITC
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
11010010001
10
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10000 signalnoiseS:N
ng antibodyIntensity
incr
easi
ng a
mou
nt o
f mAb
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.