The Keys to Successful Polychromatic Flow Cytometry

Transcription

1 Dale and Betty Bumpers Vaccine Research Center National Institute of Allergy and Infectious Diseases National Institutes of Health The Keys to Successful Polychromatic Flow Cytometry (Design and Implementation of Reagent Panels) Pratip K. Chattopadhyay, Ph.D. Staff Scientist ImmunoTechnology Section Vaccine Research Center, NIAID, NIH

2 Topics Why is polychromatic flow cytometry useful? What are the challenges to implementing this technology? are the keys to successful experiments? How do you design, evaluate, & troubleshoot panels? conjugate fluorochromes to antibodies?

3 Characterizing Cell Populations by Flow Cytometry VRC Goals Reveal T-cell subsets mediating immunity in natural disease or after vaccination. In other settings, goals differ, but usually involve cell populations that are: fundamentally important in an organism s biology, or related to disease staging or prognosis. Flow Cytometry : ideal tool for this work offers multiparametric, single cell analysis

4 What Measurements Matter? An Example from Studies of Disease Prognosis Population At-Large Infected/Diseased Protection Reduced Disease Progressive Disease The quantity (presence or absence) of a particular cell type might determine disease outcome.

5 What Measurements Matter? An Example from Studies of Disease Prognosis Population At-Large Infected/Diseased Protection Reduced Disease Progressive Disease Alternatively, the quantity may be less important than the quality. (Quality = Maturity, Function, Other Phenotypes) Polychromatic Flow Cytometry is useful because there are so many possible qualities to examine!

6 What is Polychromatic Flow Cytometry? Measurement of multiple parameters simultaneously. Why? Cells change dramatically with disease or during an immune response: e.g., T-cells: differentiation markers, cytokines, cytotoxic enzymes and we don t know how these changes relate, which predict disease, or which reflect effective immune responses. For example, a stimulated T-cell expresses multiple proteins, and only cells making certain sets are relevant to immunity. Which begs the question, how many subsets can there be?

7 Complexity of T-cell Compartment: 14-color Flow Diversity: Among memory/effector, no phenotype > 10% of cells! Over 90 phenotypically distinct populations using 6 markers.

8 So The T-cell compartment is complex...? how do we know which of these subsets is important in disease? What if the relevant cell type can only be defined by a combination of four+ markers?

9 More Colors Help Detect Correlates of Vaccine Efficacy % RA- X X X X Total CD8+ 1 marker 2 markers A+B+C+D+ A+B+C+D % RA- 57+ X X X X 3 markers 4 markers A+B A B C D A % RA X X X X A = cells expressing a certain combination of proteins (e.g., CD45RA- CD57+ CD27+ CD28+). Bulk measurements include irrelevant populations and reduce our power to detect the cells of interest. % RA X X X X Disease or Vax Efficacy

10 The Need for Polychro Flow Cellular complexity Correlates of protection lost in bulk measurements. Four colors are often inadequate. CD3 / CD4 / CD8 cells = 3 colors Dead cells introduce artifacts = 1 color Perfetto, Chattopadhyay, et al. Novel amine dyes for dead cell exclusion, JIM 2006 Naïve/Memory need 3+ markers = >3 colors Simple T-cell phenotyping = 7+ colors Chattopadhyay, et al. Immunophenotyping in HIV, Current Prot Immunology 2005 Precious samples! Why not maximize information obtained from them?

11 More Colors = More Power the rationale underlying multicolor flow cytometry.

12 More Colors Not Only = More Power More colors = more problems! (as you ll see in a moment)

13 Topics Why is polychromatic flow cytometry useful? What are the challenges to implementing this technology? are the keys to successful experiments? How do you design, evaluate, & troubleshoot panels? conjugate fluorochromes to antibodies?

14 With the right training, almost any user can do 6-8 color experiments!

15 18-color FACS not Fiction Described in: Perfetto, Chattopadhyay, et al. Nature Reviews Immunology (2004) Chattopadhyay, et al. Nature Medicine (2006) Challenges: Fluorochrome choice Reagent manufacture Panel design Quality control Data analysis <G780> <G780-A>: TNFa TNFA <B515-A>: IFN-G 105 <R710> <R710-A>: CCR7 <R660> <R660-A>: IL2 IL <G560-A>: CD154 VRC Vendors: Not all Reagent mab-fluor 16 fluorochrome Library: combinations choices. work, Roederer-inspired Over Reviewed VRC: software tools fluorochrome mab+fluor Perfetto, to suggest software Chattopadhyay, choices, best tools: combinations. in-house. FlowJo, et al. Spice/Pestle. Nature Protocols (2006).

16 Key : Separating Positive & Negative Signal What factors determine separation? Spreading Error Photon Emission Brightness of Fluorochrome. Cellular Autofluorescence PMT A Fluorochrome Related For more information, see poster 97.

17 Key : Separating Positive & Negative Signal What factors determine separation? Instrument Related PMT A Improper or Poor Quality Filters

18 Key : Well-Characterized Instruments & Reagents What factors determine separation? Fluorochrome Related Brightness PE, APC remarkably bright Cellular Autofluorescence Higher in UV/Violet Region Photon Emission Low for far red fluorochromes, so imperfect detection of low signal. Spreading Error Characteristic of staining panel, signals from other channels result in imperfect detection of low signal. Instrument Related Dynamic Range of Detector Characteristic, fixed quality Electronic Noise Laser Power Higher better Improper/Poor Filters

19 Key : Well-Characterized Instruments & Reagents What factors determine separation? Fluorochrome Related Instrument Related Brightness Dynamic Range of Detector PE, APC remarkably These bright are all things you can Characteristic, fixed quality Cellular Autofluorescence Measure Higher in UV/Violet Region Optimize Photon Emission Low for far red fluorochromes, and/or Work-Around! so imperfect detection of low signal. Electronic Noise Laser Power Higher better Spreading Error Know thy instrument, thy reagents, and Improper/Poor thyself! Filters Characteristic of staining panel, signals from other channels result in imperfect detection of low signal.

20 Topics Why is polychromatic flow cytometry useful? What are the challenges to implementing this technology? are the keys to successful experiments? How do you design, evaluate, & troubleshoot panels? conjugate fluorochromes to antibodies?

21 Developing Multicolor Panels (The Wrong Way) FITC PE-TR CD4 CD38 PE CD3 CD14 QDOT APC PE-CY5 PE-CY7 CD45RA CD154 HLA-DR A680 CD8 APC-CY7 CD57

22 Developing Multicolor Panels (The Better Way) Factors to consider: Compensation / Spreading error Level of marker expression Brightness of fluorochrome Algorithm for panel development

23 Multicolor Compensation A fact of flow cytometry life: Emission of many fluorochromes overlap. Compensation subtracts this spillover Best determined using anti-igk beads, singly-stained with each antibody, and calculated by software. (Polychro flow too complex for manual pair-wise compensation!) Quick and dirty tricks to build working panel: Use highly overlapping fluorochromes for markers that are not co-expressed (CD4/CD8). Completely avoid fluorochromes that overlap Qdots help.

24 Quick and Dirty Jump from 13- to 18-colors Organic fluorochromes: 13-color maximum. Fluorochrome overlap makes some staining resolve poorly. Quantum Dots: Inorganic nanocrystals allowed jump to 18-colors, with minimal fluorochrome overlap! Series of Quantum Dots illuminated by violet (405nm) laser.

25 Compensation Between Dots Detector QD525 QD545 QD565 QD585 QD605 QD655 QD705 QD QD QD QD QD QD Compensation between adjacent dots is fairly high. But a 5-dot system could be used where no compensation requirement is above ~10%.

26 Using Quantum Dots with Other Fluorochromes Detector QD525 QD545 QD565 QD585 QD605 QD655 QD705 FITC 7.47 PE 4.47 TRPE Cy5PE Cy5.5PE Cy7PE 3.61 APC Ax Cy7APC Almost no compensation is required from other fluorochromes. Suggests an easy strategy for panel development Construct a panel with Qdots first, and then layer on antibodies conjugated to other fluorochromes. These shouldn t affect Qdot separation.

27 But there s no such thing as a free lunch! Detector FITC PE TRPE Cy5PE Cy5.5PE Cy7PE APC Ax680 Cy7APC CasBlu QD QD QD QD QD QD Quantum Dot fluorescences appear in standard channels. FITC PE TRPE Cy5-PE, APC Cy5.5-X, Ax680 (QD525) (QD545, QD565, QD585) (QD585, QD605) (QD655) (QD705) So, the quick and dirty method to panel development has drawbacks.

28 Summary Five qdots can be used together with very little compensation. Non-Qdot fluorochromes hardly spill into Qdot channels. However, Qdot signals do appear in other, non-qdot channels. Sometimes this affects panels, sometimes it doesn t. Is it enough to simply consider compensation values? No. The picture is more complex, and depends highly on Spreading Error

29 What Is Spreading Error? When fluorescence is low, error associated with photon detection is easily observed. Particularly high photon counting errors occur for fluorochromes that give off very few photons (e.g., Cy7APC). The result: the negative population spreads out and masks dim populations in neighboring channels.

30 Know Thy Spreading Error If we calculate spreading errors for our instrument, then we ll know which fluorochrome combinations are most problematic This allows us to work around the problem of spreading error by placing dim markers on channels with lowest spreading errors.

31 Measuring Spreading Error Antibody-capture beads stained for each fluorochrome. Data collected and compensated off-line. For each fluorochrome/detector combination, spreading error is calculated. Varies instrument to instrument, must calculate for each.

32 Calculation of Spreading Error (PE bead, spread into G610)

33 FITC.fcs FLUORO Many fluoro s contribute error to PE. Still, PE is so bright, Detectors this with low doesn t error ideal matter! for dim markers Detector Qdots aren t affected by non-qdot fluoro s. 1 - FITC 2 - PE 3 - TRPE 4 - CY5PE 5 - CY55PE 6 - CY7PE 7 - APC 8 - AX CY7APC 10 - CBLUE 11 - QD QD QD QD QD QD655 PE.fcs TRPE.fcs Cy5PE.fcs Cy55PE.fcs Cy7PE.fcs APC.fcs Ax680.fcs Cy7APC.fcs CBlue.fcs QC525.fcs QD545.fcs QC565.fcs Total Error Recv d 17 - QD705 Fluorochrome Total Error Contributed QD655 contributes SE into Cy55PE but not TRPE QC585.fcs QC605.fcs QC655.fcs QC705.fcs

34 Review : How to Develop A New Panel Don t randomly combine antibodies and fluorochromes. Quick & Dirty The Better Way Pick fluorochromes that don t overlap. Qdots are particularly useful for this. Pair highly overlapping fluorochromes with markers that aren t co-expressed. Characterize spreading error for the fluorochromes and instruments you use. Place dim markers on channels with low spreading error. Continue panel development using the following example

35 Designing a Multicolor Panel Considerations: 1. What do you want to identify? Minimum set of necessary markers Multiple panels vs. single panel 2. What do you want to exclude? Dump channel Negative markers 3. What additional markers might you use? Rank: Is it useful, or is it luxury?

36 How Many Markers to Use? Always tempting to use as many markers as possible. However, remember: More Colors = More Power = More Problems Problems include: Loss of sensitivity Spreading error usually increases w. more fluorochromes. Unwanted FRET Reagent interactions Rare, but possible.

37 How Many Markers to Use? Classify reagents into three groups: (1) Absolutely necessary (2) Important (3) Luxury Then consider the characteristics of the fluorochromes available for your instrument. Finally, consider the expression patterns of the markers you will measure.

38 Consider Fluorochrome Characteristics All colors are not created equal. Anti-CD8 FITC, PE, Cy5PE, APC, Cy7APC can show different distributions on singly-stained cells. Two facets contribute to this: Reagent brightness: Compared to autofluorescence, dimly stained cells may resolve with some colors but not others (combination of brightness, AF, sensitivity) Absolute signal: PE yields many more photons per antibody-conjugate than Cy7PE, hence the width (CV) of distributions is narrower, providing better separation even for brightly-stained cells.

39 Consider Expression Patterns We divide reagents into three categories: Primary Well-characterized, identify broad subsets of cells, expression is usually on/off. e.g., CD3, CD4, CD8, CD14, CD19, CD20 Typically used as parent gates in analysis Secondary Well-characterized, bright expression patterns e.g., CD27, CD28, CD45RA/RO, γifn, perforin Expression levels can be a continuum Tertiary Low-expression levels or uncharacterized e.g., CD25, CCRs, X

40 Selection of Marker/Color Combinations Tertiary Low-expression levels or uncharacterized e.g., CD25, CCRs, X These require the absolutely brightest colors, with the least spillover problems possible e.g. PE, APC, QD655

41 Selection of Marker/Color Combinations Secondary Well-characterized, bright expression patterns e.g., CD27, CD28, CD45RA/RO, γifn, perforin Expression levels can be a continuum These are usually assigned to the next tier of colors, those that perform well with little spillover problems e.g., FITC, TRPE, Cy5PE/PerCP, Alexa 405, Alexa 680

42 Selection of Marker/Color Combinations Primary Well-characterized, identify broad subsets of cells, expression is usually on/off. e.g., CD3, CD4, CD8, CD14, CD19, CD20 Typically used as parent gates in analysis These reagents are usually assigned to dimmer fluoros and colors that exhibit the greatest spillover problems e.g., Cy5.5PE, Cy7PE, Cy7APC, QD800

43 Planning a Panel FLUOROCHROME MARKER CATEGORY FITC CD28 LUXURY PE TETRAMER TERTIARY TRPE CCR7 LUXURY CY5PE PI PRIMARY CY55PE CY7PE CD14 PRIMARY APC TETRAMER TERTIARY A680 CD4 PRIMARY CY7APC CD3 PRIMARY CBLUE CD57 SECONDARY Q545 Q565 CD8 PRIMARY Q585 Q605 CD27 SECONDARY Q655 CD95 LUXURY Q705 CD45RA SECONDARY Q800

44 Planning a Panel FLUOROCHROME MARKER CATEGORY FITC CD28 LUXURY PE TETRAMER TERTIARY TRPE CCR7 LUXURY CY5PE PI PRIMARY CY55PE CY7PE CD14 PRIMARY APC TETRAMER TERTIARY A680 CD4 PRIMARY CY7APC CD3 PRIMARY CBLUE CD57 SECONDARY Q545 Q565 CD8 PRIMARY Q585 Q605 CD27 SECONDARY Q655 CD95 LUXURY Q705 CD45RA SECONDARY Q800

45 Example Optimization Goal: to evaluate the expression of CXCR3 and CCR4 on naïve (CD62L + CD45RA + CD45RO ) CD4 T cells. What fraction of naïve T cells express these molecules? If possible: are those cells truly naïve (CD28 + CD11a dim CD27 + )? Requirements: CD4, CD3 = Primary reagents CD45RO/RA, CD62L = Secondary (need excellent separation) CXCR3, CCR4 = Tertiary reagents CD27, CD11a, CD28 = Luxury reagents

46 General Approach 1. Assign tertiary reagents to best channels (FITC, PE, in this case). 2. Test all conjugates of Secondary reagents to determine how good they are. 3. Choose 3-4 best conjugates, and construct panels with Primary reagents slotted in. 4. Evaluate expression patterns to ensure appropriate identification of naïve/memory subsets. 5. Evaluate potential sensitivity of FITC and PE channels (where CXCR3 and CCR4 will be used).

47 CD45RO Example Stains : Various Conjugates For panel

48 Design of Panels Need optimal sensitivity, therefore tried to minimize reagents that would spillover/spread into FITC and PE. Optimal separation of CD62L and CD45Rx was required. Other memory markers less important: therefore, some panels tested minimal requirements, and others were part of the wish list.

49 First Set of Panels TRPE Cy5PE Cy55PE Cy7PE APC Cy55APC Ax680 Cy7APC CB QD655 1 CD45RA CD4 CD27 CD62L CD11a CD45RO CD3 2 CD45RO CD4 CD27 CD45RA CD11a CD62L CD3 3 CD45RO CD45RA CD62L CD27 CD4 CD11a CD3 4 CD45RA CD62L CD4 CD45RO CD3 5 CD62L CD4 CD45RA CD45RO CD3 6 CD45RA CD11a CD27 CD62L CD4 CD45RO CD3 7 CD4 CD45RA CD62L CD27 CD28 CD11a CD45RO CD3 8 CD45RO CD3 CD62L CD28 CD11a CD4 CD27 CD45RA

50 Panel Evaluation: First criterion: RO vs. 62L Cy55APC CD62L: Too much smearing in some panels. CD45RO: Looks good in all panels

51 Third criterion: Sensitivity for FITC, PE FITC channel not affected much. PE strongly affected what is causing this? Note: The importance of FMO controls in gating!

52 Second Series of Panels Based on the evaluation of the first set of panels, certain combinations were eliminated. The good aspects of other combinations were combined and fine-tuned. TRPE Cy5PE Cy55PE Cy7PE APC Cy55APC Ax680 Cy7APC CB QD655 9 CD3 CD62L CD4 CD45RO CD45RA 10 CD45RO CD3 CD62L CD28 CD11a CD4 CD27 CD45RA 11 CD45RO CD3 CD28 CD62L CD4 CD27 CD45RA 12 CD45RO CD4 CD27 CD45RA CD11a CD62L CD3 Panel 9: Aim for maximal sensitivity in FITC, PE

53 Final Panels TRPE Cy5PE Cy55PE Cy7PE APC Cy55APC Ax680 Cy7APC CB QD655 1 CD62L CD4 CD45RO CD45RA 2 CD45RO CD3 CD62L CD28 CD11a CD4 CD27 CD45RA 3 CD45RO CD27 CD4 CD11a CD62L CD45RA Note: CD3 was dropped from 1 & 3 as CD4 staining was deemed good enough to identify CD4 T cells. Panel 2 will validate this assertion! Panels 2 & 3 add more memory markers to verify the final phenotype of the chemokine-expressing cells.

54 Result Final panel worked very well--in fact, identified expression of CCR4 not previously seen on FACSCalibur!

55 Is a long, complicated, iterative process. Plan to spend 5 experiments minimum. (1): Survey range of reagents Panel Optimization (2): Construct 8-12 possible multicolor combinations (3): Rank each combination, deriving rules about reagents and combinations. Construct 4-6 derivative combinations (4): Repeat step 3, winnowing down the combinations. Record the process as you go along!

56 Chattopadhyay, Nature Medicine, August 2006

57 Antibody Conjugation Basic needs Types of conjugations Procedures Validation See:

58 Basic Needs Bulk purified unconjugated antibody NO EXOGENOUS PROTEIN Concentrated (>4 mg / ml) Reactive fluorescent reagents FITC, Alexa x, TR: Molecular Probes PE, APC: ProZyme, Molecular Probes, others Cy5, Cy5.5, Cy7: Amersham (Need agreement) Desalting columns (e.g., PD-10), buffers, tubes, dialysis equipment

59 Conjugation Types Simple (purchase reactive dye): FITC, Alexa-X, Cy-X, TR (usually). mw ~ 1,000. Intermediate (prepare reactive dye): PE (240 KDa), APC (104 KDa) (Note: Kits available to make this simple!) Complex (optimize conditions, prepare tandem, activate to prepare reactive dye): TRPE, Cy5PE, Cy5.5PE, Cy5.5APC, Cy7PE, Cy7APC (mw similar to PE or APC)

60 Antibody Preparation Need bulk purified concentrated antibody Purchase from vendor (request special preparation): typically must purchase 5-10 mg quantities to get decent price; will cost US$2,500 to $6,000 per 10 mg. Prepare from hybridoma: Use BD CellLine flask to grow; once cells are growing, requires 3 week culture (very simple); purify over simple thiophyllic column from Pierce; yield up to 150 mg. Primary cost: labor plus US$ Prepare from ascites: Primary cost: animals, labor. Purify over protein G or simple thiophyllic column; yield up to 150 mg.

61 Antibody Preparation Purified antibody can be stored frozen, although recommend storing at 4C in buffer or as ammonium sulfate precipitate. Prior to conjugation, antibody must be prepared: (1) If precipitate: dialysis extensively into buffer (2) If frozen: centrifuge at high speed (100,000 x g) to remove aggregates (3) For protein-protein conjugations: reduce disulfide intrachain link with DTT (4) Exchange over column into appropriate reaction buffer.

62 Simple Conjugations FITC, Alexa-X, TR, Cy-X: Purchase reactive dyes: fluorescent reagent has highly reactive group that will react with primary amines (lysine). Reconstitute small amount of reactive dye in DMSO (single-use only!). Add reactive dye to antibody; react for 1-4 hours; exchange over desalting column (PD-10) into storage buffer. DONE!

63 Simple Conjugations FITC, Alexa-X, TR, Cy-X: P.S. Typically, This is will one use reason 3x to isotype 20x molar controls excess aren t over antibody really controls. (final, 3-5 The dye optimization molecules per of antibody). F/P ratio may differ for the In isotype general, and you the should specific optimize antibody! reaction conditions on a prototype antibody. Carefully define ph of buffers! Brightness F/P Optimum More nonspecific binding

64 Simple Conjugations FITC, Alexa-X, TR, Cy-X: Note: In some cases, simple conjugation may destroy antibody reactivity. This is fairly common for TR. You can try alternative reactive groups. Or (nearly always works): Conjugate BSA with reactive dye (5-20 molar excess). Prepare BSA for proteinprotein conjugation; conjugate fluorescent BSA to antibody through hinge-disulfide.

65 Protein-Protein Conjugations PE, APC: Antibody is reduced with DTT to expose hinge disulfide. PE or APC is activated with SMCC in a nearly identical reaction as FITC on antibodies: 3-10x molar excess (optimization is useful). SMCC-PE or SMCC-APC is added to antibody at 2-4 molar excess, for 1 hour. Add N-ethylmaleimide (NEM) to block free -SH and quench reaction. Exchange over desalting column into storage buffer.

66 Protein-Protein Conjugations PE, APC: ProZyme sells kit with pre-activated PE or APC (US$ per kit; sufficient to conjugate 1 mg of antibody; can split across multiple antibodies if you supply additional columns). Reactive PE or APC can be prepared in bulk (e.g., 100 mg) and stored for years in active form. Purchase PE or APC at bulk: 1g for US$5,000-10,000. Final cost: $10-20 per mg of antibody.

67 Post-Conjugation Before final desalting column to exchange protein into storage buffer, centrifuge at 10,000 x g for 10 min to remove aggregates. Routinely centrifuge conjugates (monthly) before use. Re-titrate every year. Occasionally, some conjugates are not stable (less than 1 in 20).

68 Post-Conjugation Is purification necessary? For FITC, CyX, Alexa: No. Final desalting column removes unreacted dye. 3:1 to 10:1 reaction ratio ensures no unreacted antibody. For Protein-protein: In general, no. 2:1 reaction ratio ensures no unreacted antibody. 50% of material is unreacted protein dye, which washes away after surface staining. However, for Intracellular staining, Yes. Use Sephadex 400 sizing column. Lose 30-50% of material; laborintensive.

69 Post-Conjugation VALIDATE ALL REAGENTS. Confirm specificity. Blocking Experiments Compare to commercial reagents. Titrate. Titrate. Titrate. Re-validate on annual basis, or as necessary.

70 So far Characterized Instrument Built Panels Optimized Panels Produced New Conjugates

71 Next : Evaluating the Panel How do you test whether panel is working? Once preliminary gating is complete (i.e., excluding dead cells, identifying lymphocytes), examine every combination of markers. For example, if the panel consists of five reagents (A-E), plot A vs. B, A vs. C, A vs. D, A vs. E. Next: B vs. C, B vs. D, B vs. E. Then: C vs. D, C vs. E, and D vs. E. Flag suspicious staining patterns.

72 Flag Suspicious Patterns Very little Ki-67 HLA-DR biology? Retitrate Poor CD69 Leaner Poor CD38 Very little CD25

73 Develop an Action Plan for Each Problem Very little expression: Examine different subset (not expressed in CD4, but what about CD8?) Try different sample, try stimulation. Biologically questionable: Test smaller panel on same sample, compare against commercial reagent, examine other marker combinations to see if reason can be identified. In last example, further investigation showed that HLA-DR had bright and dim populations because CD14+ cells were not gated out. Too much background: Re-titrate antibody. Poor separation: Examine different subset, compare to commercial reagent, re-design panel considering factors addressed today. Leaning populations (of Pisa): Suggest compensation problems. Re-run comp tubes or experiment.

74 Other Examples of Potential Problems Biologically impossible: Lots of cells double positive for markers that should rarely be co-expressed. Fluorochrome aggregates: Usually not a problem if events are few and scattered, just gate them out. When lots of agg, big reagent problem. Diagonal populations: Highly correlated expression is rare. Think through whether it is biologically possible, or compensation error. Leaners: Suggest compensation problems. Negative population too bright, or all cells positive: Re-titrate reagent. Too little expression, or poor separation: Compare to another reagent, re-do experiment (just to see if it repeats), simplify panel and build it again.

75 Once Satisfied with Panel Avoid changing reagent lots (especially of in-house conjugates) in the middle of a large study. Test it a few times before undertaking large study. See if it holds up when you stain multiple samples at once, or in a plate. Do a dry run of study conditions (without precious samples) before large studies. Have a means to check panel in every experiment. I keep a wellcharacterized control, or simply a sample of fresh healthy donor cells, to run each experiment day. Use movies to rigorously check staining patterns and gating between samples and between experiment days.

76 Movie Time!

77 Summary Polychromatic flow cytometry and panel development is not easy There are lots of things to consider, but you ll be fine if you: Start with a reliable, well-characterized system Are methodical in the steps you take for panel development Consider increasing your reagent flexibility with in-house conjugates Use proper controls (e.g., FMOs) Always critically evaluate the data you are generating and perform sacrificial offerings to the flow cytometry Gods. :)

78 Acknowledgements My work in polychromatic flow wouldn t be possible without fantastic mentorship: Mario Roederer Steve Perfetto Steve De Rosa without many, many great collaborators, both for studies of disease in the various areas of biology, and technology, and without the tools provided by lots of top-notch commercial vendors.

79 Final Notes Comments? Questions? Please pchattop@mail.nih.gov This material is provided as a service to the flow cytometry community. Please do not re-package elements of this presentation or copy slides without prior consent and proper attribution.