Flow Cytometry: From Basic Concept To Advanced Sorting 파멥신 / 한국화학연구원 김중규

Transcription

1 Flow Cytometry: From Basic Concept To Advanced Sorting 파멥신 / 한국화학연구원 김중규

2 Contents 1. Basic Introduction and Application of Flow Cytometry 2. Choosing Antibody, Compensation, and Controls 3. Sorting 4-1. Basic principles of cell sorting 4-2. Advanced understanding of sorting 4-3. Sample prep and other things influencing sorting

3 Properties of FSC and SSC Side Scatter (SSC) : Granularity or Internal Complexity LASER Forward Scatter (FSC) : Size SSC Granulocytes Monocytes Lymphocytes FSC

4 Hydrodynamic focussing in the cuvette (fluidics) Sample Sheath Sample Sheath Sample pressure low, small core stream. Good for DNA analysis High sample pressure, broader core stream. Bad for DNA analysis LOW HIGH

5 Using Multi-Laser: Time delay matters

6 Examples of optical filters in flow cytometry Long pass(lp) Short pass(sp) Band pass(bp) LP 500 SP 500/50

7 Electronics Laser Voltage Pulse analysis Time Laser Laser Voltage Pulse FSC SSC Voltage Voltage Time Time Pulse Height Pulse Width Pulse Area Data processing SSC PE FSC FITC Time Time APC FL1 Time FL2 Time PerCP-Cy5.5 FL3 FL4 Time Time

8 Application of Flow Cytometry Surface phenotyping : Ab against cell surface Ag, MHC tetramer etc Staining of intracellular protein after cell permeabilization ICS (intracellular cytokine staining) with brefeldin A/monensin Detection of phosphorylated protein Cell proliferation (CFSE, BrdU etc) Analysis of intracellular ion concentration Analysis of reduction/oxidation (redox) potential Cell cycle analysis (by using DNA-binding dye) Apoptosis analysis (annexin V, TUNEL etc) Cytokine secretion assay (& subsequent isolation) CBA (cytometric bead array) - Multiplex proteins assay Sorting

9 Western vs Flow Cytometry

10 In the case of therapeutic Ab development, why we need flow cytometry?

11 mrna and IHC info shows different results, and not compatible with FACS data (ex. MDA-MB-231)! KDR DLL4 Notch1 EGFR U87MG_CNS MDAMB231_BREAST MCF7_BREAST COLO205_COLON KDR DLL4 Notch1 EGFR A549_LUNG From Broad-Novartis Cancer Cell Line Encyclopedia (CCLE) HUVEC MDA.MB.231 KDR Notch1 Notch1 EGFR higg Tanibirumab Isotype EGFR DLL4 Notch1

12 Contents 1. Basic Introduction and Application of Flow Cytometry 2. Choosing Antibody, Compensation, and Controls 3. Sorting 4-1. Basic principles of cell sorting 4-2. Advanced understanding of sorting 4-3. Sample prep and other things influencing sorting

13 Three Categories for Antigens 1. Primary Ags : On/Off (e.g. CD3, CD4, CD8, CD14, CD19,CD20..) 2. Secondary Ags : Continuum (e.g. CD27, CD28, CD45RA/RO, IFN-g, Perforin..) 3. Tertiary Ags : low level or low population, (e.g. CD25, Chemokine receptors) or unknown/most important Ag Basic rule for Ab combination Brighter Abs for Tertiary > Secondary > Primary

14 Brightness = Resolution Sensitivity Stain Index = D / W Where D = difference between positive and negative peak medians W = 2 x rsd (robust standard deviation) Holden Maecker & Joe Trotter, Nature Methods 5, (2008)

15 Antibody Titration signal noise 1000 S:N ng antibody 1

16 Let s think about Ab concentration! You are staining CD4 (100,000/cell) on 1 x 10 6 cells. You are using anti-cd4 Ab at 2ug/ml conc., and the staining volume is 0.1ml How many anti-cd4 antibodies are there for one CD4 molecule?

17 Spillover also affects resolution sensitivity Without CD45 AmCyan: With CD45 AmCyan: CD19 FITC Note that this is only an issue when the two markers (CD45 and CD19) are co-expressed on the same cell population.

18 Compensation Laser 488 nm FITC PE To compensate FITC overlap in FL2 Subtract x% of FL1 from FL2 To compensate PE overlap in FL1 Subtract x% of FL2 from FL1

19 Compensation - too little, too much

20 Three categories of controls 1. Instrument set-up and validation controls (commercial) 2. Gating controls including FMO(Fluorescence-Minus-One) 3. Biological controls e.g. co-stimulation only All controls except commercial beads should be treated as same as the experimental samples e.g. fix/perm, incubation

21 Use of FMO (Fluorescence Minus One) controls Perfetto SP, Chattopadhyay PK, Nat Rev Immunol. 2004

22 Contents 1. Basic Introduction and Application of Flow Cytometry 2. Choosing Antibody, Compensation, and Controls 3. Sorting 4-1. Basic principles of cell sorting 4-2. Advanced understanding of sorting 4-3. Sample prep and other things influencing sorting

23 Sorting FACSAria system Flowcell Cuvette Nozzle Deflection Plates Collection Tubes

24 What is drop delay? Cuvette Point of analysis Nozzle Drop Delay Sort pulse (Drop Charge)

25 Practically important points Sort FAST Sort PURE Sort with a high YIELD

26 Sort speed Sorting speed is dependent on fluid pressure and nozzle size Example: At 70 psi preessure, using a 70 µm nozzle, the frequency of drop formation can be up to ~90 khz. I.e drops per second can be formed. Drop formation frequency ~ Jet velocity Jet Velocity ~ Pressure KHz would require 500 psi (!)

27 Purity the reality AND! AND!

28 Purity - gating strategy for singlet, live target cells * Mahnke YD, Roederer M, Clin Lab Med., 2007

29 Contents 1. Basic Introduction and Application of Flow Cytometry 2. Choosing Antibody, Compensation, and Controls 3. Sorting 4-1. Basic principles of cell sorting 4-2. Advanced understanding of sorting 4-3. Sample prep and other things influencing sorting

30 What can I expect from a sorter? Depends on what you want: Purity Yield Count accuracy Viability Speed!!

31 Sorting in short Cell sorters cannot sort cells, they sort droplets They always sort the last attached droplet You better make sure the cells of interest are in those droplets And, if you want purity, that no others are in there Catch all of them in the appropriate tube Count them correctly What you find in your tube is exactly what you asked for And some cross-contamination if you don t take care

32 Purity and sort coincidence Purity is also maintained by detecting sort coincidence. Sort coincidence two or more particles are too close together to be separated in two individual drops

33 Sort decisions A sort decision must be made for each particle The sort decision is based on the sort gate AND sort mask. Sort masks can be defined based on the purpose of the sort. There is a tradeoff between purity and yield. The effect of this tradeoff is dependent on sort speed and the frequency of the cells of interest.

34 Yield mask Yield mask - to minimize loss of cells due to position uncertainty: Two drop sort

35 Purity mask Purity Mask - to avoid contamination: No sort contaminating cell in purity mask

36 Phase mask Phase Mask - to ensure count accuracy: Sort

37 Sort envelop: 32yield32purity0phase Sort Sort

38 Sort envelop: 32yield32purity0phase Sort Sort

39 Summary Yield mask to recover cells that would be lost due to position uncertainty Purity mask to avoid contamination Phase mask for 100% count accuracy

40 Speed Purity Yield tradeoff The purity/yield tradeoff increases in importance with increasing sort speed

41 Speed Purity Yield tradeoff (ideal) Hz ev/s Hz ev/s

42 Speed Purity Yield tradeoff (real) Hz ev/s Hz ev/s

43 Conflict Resolution Event Rate (events/sec) No. Particles 1,000 10,000 20,000 30, % 82% 67% 55% 1 2% 16% 27% 33% 2 0% 2% 5% 10% 3 0% 0% 1% 2% 4 0% 0% 0% 0% 5 0% 0% 0% 0% % 1 particle Table 1. Probability of having n particles in a drop for four different event rates at a drop frequency of 50 khz

44 Contents 1. Basic Introduction and Application of Flow Cytometry 2. Choosing Antibody, Compensation, and Controls 3. Sorting 4-1. Basic principles of cell sorting 4-2. Advanced understanding of sorting 4-3. Sample prep and other things influencing sorting

45 Basic considerations for Ab staining Most Abs bind well at RT or 4 o C, for 10-15min. In some cases, 37 o C more preferable, but be sure that the cells are rather active at this temperature than RT or 4 o C Chemokines and their receptors exceptionally sensitive to variations in cell isolation and staining techniques Do not ignore the specified/complicated staining protocols for some special cases they deserve it. But if you want to test, go ahead for your efficiency.

46 Additional considerations for sample preparation Dead cell discrimination necessary - DNA-intercalating dyes (EMA, PI, 7-AAD, DAPI) - Phosphatidylserine-binding reagents (annexin V) - Amine reactive dyes (UViD, ViViD, GrViD, OrViD) Qdot and tandem dye(pe-cy5.5, PE-Cy7, PerCP-Cy5.5 ) be aware of aggregation and decay, respectively! Consider specific situation - e.g, CD3 down-regulation upon T-cell stimulation Be meticulous! It will eventually save a lot of your time and energy!

47 References Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Bendall et al. Science May 6;332(6030): Flow cytometry, amped up. Benoist and Hacohen. Science May 6;332(6030):677-8 A flow cytometry revolution. Doerr A. Nat Methods Jul;8(7):531. Optimizing a multicolor immunophenotyping assay. Mahnke and Roederer. Clin Lab Med Sep;27(3): Modern flow cytometry: a practical approach. Tung et al. Clin Lab Med 2007 Sep;27(3): Flow cytometry controls, instrument setup, and the determination of positivity. Maecker and Trotter Cytometry A 69: Seventeen-colour flow cytometry: unravelling the immune system. Perfetto et al Nat Rev Immunol. Aug;4(8): Selecting fluorochrome conjugates for maximum sensitivity. Maecker et al Cytometry A 62: 169. And various online sites including BD, Stanford, Purdue university, NIH

48 Thanks!