Introduction into Flow Cytometry Stephanie Gurka, Andreas Hutloff, Timo Lischke, Kroczek-Lab, Robert Koch Institute, Berlin
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 Use of fluorescence marked monoclonal antibodies multi-parameter analysis (up to 18) for each individual cell High flow-rate (> 20,000 cells/sec)
Particles can be measured with a flow cytometer Blood cells Tissue cells Algae Protozoa Chromosomes Yeast Prerequisite: single cell suspension (Disaggregation: mechanical or enzymatic)
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 time delay 488 nm Laser 633 nm Laser 55 ms 17 ms 405 nm Laser
Optics - Forward Scatter Channel (FSC) Coherent lightsource (488 nm) detect the amount of light scattered in the forward direction (along the same axis that the laser light is traveling)
voltage voltage voltage Forward Scatter Detector time particle passes through the focus -> scattered light is detected by a photodetector, -> an electrical pulse is generated and presented to the signal processing electronics.
Intensity of forward scatter is most influenced by the size of cells 10 FSC-Histogram 9 Cell count 8 7 6 5 4 3 2 1 0 Intensity
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
Optics - Side Scatter Channel (SSC) detect the amount of light scattered to the side (90 to the axis that the laser light is traveling) Intensity of SSC is most influenced by the shape and optical homogeneity of cells
Scatter Plot SSC tends to be more sensitive to inclusions within cells -> can be used to distinguish granulated cells from non-granulated cells
Which parameters can be measured? the relative size (FSC) the relative granularity or complexity (SSC) the fluorescence intensity (FL1/2, up to FL X) -> 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 530nm FITC Stokes Shift 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 Excitation/Emission Spectra
Fluorescence detection Fluorescence emitted by each fluorochrome is usually detected in a unique fluorescence channel. Fluorescence detector (PMT3, PMT4 etc.) SSC detector photo multiplier tube (PMT) FCS detector photo diode Laser Specificity controlled by the wavelength selectivity of optical filters and mirrors. signal levels are high
Optical Layout - BD FACSCalibur
Resolution Sensitivity: Clone RPA-T4 D W1 W2 Stain Index = D / W 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 D = difference between positive and negative peak medians W = 2 x rsd (robust standard deviation)
Fluorescence One Color Histogram in conjunction with fluorescence-based protein reporters (GFP) -> monitor both transfection efficiency and protein expression levels.
Data Analyis cell count Histogram: Light/Fluorescence Intensity Intensities of 2 Light/Fluorescence Parameters plotted against each other CD8 2D plot: CD4
Gating - Statistics MFI =10 Mean Fluorescence Intensity (MFI) Quadrant Statistic % Y+X- % Y+X+ MFI =150 %A Y %C %B % Y-X- % Y-X+ X
Gating - example
Gating - example
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.
Fluorescence dyes used for flow cytometry http://www.bdbiosciences.com/spectra/ Fluorochrome excitation wavelength (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 Company FITC, PE, PerCP, APC, Cy5 Becton Dickinson Alexa Fluor Molecular Probes (Invitrogen) efluor ebioscience V BD Horizon Pacific Oregon
Quenching Relative intensity Fluoresence intensity FITC ph dependence Concentration fluorescein ph Wanted properties of fluorochromes Brightness (high quantum yield) Photostability (no bleaching) ph insensitivity (stability of fluorescence emission) Water solubility (little hydrophobic interactions) % inital fluoresence intensity Fluorescence: points to consider Photobleaching Photostability / Photobleaching Instrument compatibility (fit to excitation wavelenght) Multiparameter Time (sec) (small emission spectrum) Molecular Probes
Types of Fluorochromes I) Small dyes: FITC, Cy5, AlexaFluor s, efluor s, FITC I) I) g Large Protein dyes: phycoerythrin, allophycocyanin, peridinin-chlorophyll-protein PEB accessory photosynthetic pigment of red (R-PE, PerCP) or bluegreen algae (APC). PE: APC: PerCP: 240-kDa protein with 34 phycoerythrobilin fluorochromes per molecule. 105-kDa protein with 6 phycocyanobilin chromophores per molecule. 35-kDa protein with phycoerythrobilin fluorochromes
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
Coupling of fluorescent dyes to antibodies II) Protein dyes (phycoerythrin, allophycocyanin) Amine - thiol crosslinking 1) The bifunctional crosslinker Succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1carboxylate (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
Antibody titration basics 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 increasing amount of mab 10000 signal noise S:N 1000 100 10 Intensity 1 1000 Titer is affected by: Staining volume Number of cells Staining time and temperature Type of sample (whole blood, PBMC, etc.) 100 10 ng antibody 1
Fluorescence Tandem
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 positive population negative population Filters collect 2 colors
Fluorescence Compensation positive population negative population 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
Compensation controls 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 Small errors in compensation of a dim control can result in large compensation errors with bright reagents CompBeads manually (up to 4 FL) or automatic compensation (>4 FL)
Specificity Controls Controls must undergo the same treatment (i.e., preparation, fixation) as all the tubes in an experiment. 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
Further Specificity Controls 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.
Comparison of gating controls
Doublet discrimiation based on fluorescence height, fluorescence area and signal width.
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.
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 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.
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 Isotype Control 13.68 75.29 26.84 28.20 FITC PE PE-Cy7 APC-Cy7 a- hu CD4 conjugates
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 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)
Analytical Variables to consider Sample preparation time, temperature, buffer (ph, salt concentration) Lysis, digestion, fixation, permeabilisation, washing steps instrument number and type of Lasers, Filters, Fluorescence Detectors -> antibody fluorochromes/ -combinations clone, affinity, monoclonal vs. polyclonal, Ig-Isotype, type of Fluorochrome, concentration, F/P ratio, (may differ from lot to lot) staining procedure: 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 Rapid and scalable: Performed in 96-well plates and in parallel
Multicolor analysis: Choice of Ab-Fluorochrome 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 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.
Analytical Variables to consider Data Acquisition, Analysis and Interpretation Instrument setup and performance adjust and optimize PMT settings (optimal sensitivity) Set voltages: Decrease voltages for any detectors where events are off-scale Increase voltages for any detectors where low-end resolution is poor 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) Speed of analysis (high flow rate -> less intensity resolution)
Analytical Variables to consider 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
Rare Event Detection 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
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.