Best practices in panel design to optimize the isolation of cells of interest

Transcription

1 Sort Best practices in panel design to optimize the isolation of cells of interest For Research Use Only. Not for use in diagnostic or therapeutic procedures. Alexa Fluor is a registered trademark of Life Technologies Corporation. Cy is a trademark of GE Healthcare. Cy dyes are subject to proprietary rights of GE Healthcare and Carnegie Mellon University and are made and sold under license from GE Healthcare only for research and in vitro diagnostic use. Any other use requires a commercial sublicense from GE Healthcare, 800 Centennial Avenue, Piscataway, NJ , USA. Trademarks are the property of their respective owners BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. The BD Horizon Global Tour

2 Cell sorting Cell sorting: isolation of a population of interest for downstream analysis Cell enrichment Cell transplantation Downstream functional and genomic analysis Goal: to obtain a pure population with maximum yield The BD Horizon Global Tour 2

3 Increasing resolution for cell sorting Clear resolution of a population of interest is critical for an optimal sort. How to increase resolution in a sorting setting? Eliminate the impact of unwanted cells Increase the ability to visualize from population of interests. The BD Horizon Global Tour 3

4 CD3 Increasing resolution for cell sorting Clear resolution of a population of interest is critical for an optimal sort. PE PE-CF594 BV421 Alexa Fluor 647 PerCP-Cy 5.5 Alexa Fluor 700 V450 FITC CD197 (CCR7) How to increase resolution in a sorting setting? Eliminate the impact of unwanted cells. Increase the ability to visualize population of interests. The BD Horizon Global Tour 4

5 Considerations for cell sorting Cell analysis Fluorochromes Biology Instrument setup Cell sorting Fluorochromes Biology Instrument setup Sample preparation Gating strategy The BD Horizon Global Tour 5

6 Considerations for cell sorting: overview Experiment setup Sample preparation Instrument settings Gating strategy Histogram vs plots Biexponential scale Doublet discrimination Data display Panel design Dead cell exclusion Lineage exclusion/depletion Antibody titration Fluorochrome choice The BD Horizon Global Tour 6

7 Considerations for cell sorting Sample preparation, instrument setup The BD Horizon Global Tour 7

8 Sample preparation Cell dissociation/detachment Cell resuspension buffer Staining volume and antibody concentration Cell sorting buffer and cell density Optional use of DNAse Temperature, ph Sample collection: Cell collection buffer (cell culture, transplantation, genomic analysis) Temperature, ph The BD Horizon Global Tour 8

9 Instrument settings Nozzle size/sheath pressure 70 µm/70 psi for lymphocytes 100 µm/20 psi for larger and/or fragile cells Event rate (number of events/second) Low speed for higher sorting efficiency Sort setup Bulk sorting Purity vs yield Single cells Laser alignment Drop delay The BD Horizon Global Tour 9

10 Instrument settings: speed vs yield vs purity Influenced by: Drops per second, events per second, sort mask and target population frequency Speed Percent Yield Events/second (x 1000) Purity Yield Good rule of thumb: Maximum recommended event rate = drops per second / 5 The BD Horizon Global Tour 10

11 Considerations for cell sorting Gating strategy Histogram vs plots, biexponential scale, doublet discrimination, data display The BD Horizon Global Tour 11

12 Count CD-Y CD-Y Different options for data display Histogram Dot plot Contour plot CD-X CD-X CD-X The BD Horizon Global Tour 12

13 Count SSC-A Count SSC-A Histograms vs plots: How many populations do you see? CD-X BUV395 CD-X BUV395 CD-X BUV CD-X BUV395 CD4 BUV395 The BD Horizon Global Tour 13

14 CD45RA APC CD45RA APC Dot plots vs contour plots: How many population do you see? CD197 BV421 CD197 BV421 The BD Horizon Global Tour 14

15 Count SSC-A Histograms vs bivariate plots: where to draw the gate? GFP GFP Transfected cells express different levels of GFP. Bivariate plots better reveal the separation from negative/dim to positive cells. What if GFP is expressed at low levels? The BD Horizon Global Tour 15

16 SSC-A SSC-A Gating low GFP expressing cells: what is real and what is not? Parental cells Transfected reporter cells GFP GFP High background in the GFP channel is usually due to autofluorescence. Negative controls are instrumental for proper gating. The BD Horizon Global Tour 16

17 SSC-A SSC-A Gating low GFP expressing cells: what is real and what is not? Parental cells Transfected reporter cells 2% 3.8% GFP GFP High background in the GFP channel is usually due to autofluorescence. Negative controls are instrumental for proper gating. The BD Horizon Global Tour 17

18 PerCP-Cy5.5 PerCP-Cy5.5 SSC-A SSC-A Gating low GFP expressing cells: leveraging autofluorescence Parental cells Transfected cells 2.5% 4.8% Parental cells Transfected cells 0% 3.1% GFP PerCP-Cy5.5 GFP GFP Autofluorescence is detected in multiple channels. Plot GFP against another channel with autofluorescence. Autofluorescent cells will be double positive (diagonal), revealing true GFP single positives.

19 The biexponential scale: the best way to look at compensated data Standard log scale This population looks under compensated. These look like two separate populations. Visualization of compensated data is greatly improved using the biexponential scale. 1x <PE-A> Biexponential scale This example showing different displays of the same data shows the value of the biexponential scale, a mostly logarithmic scale on the upper end, linear at the low end and symmetrical about the negatives. Compensated single positives are continuous. All populations are visible x10 5 <FITC-A> The BD Horizon Global Tour 19

20 CD3 BUV395 CD3 BUV395 CD3 BUV395 Gating strategy: biexponential scale CD4 PerCP-Cy5.5 CD4 PerCP-Cy5.5 CD4 PerCP-Cy5.5 The BD Horizon Global Tour 20

21 Doublet discrimination Sort check The BD Horizon Global Tour 21

22 Signal Signal Signal Electronic pulse Time Time Height Area The BD Horizon Global Tour 22 Time Width

23 Signal Signal Doublet discrimination Time 1x Width The BD Horizon Global Tour 23 Time 2x Width

24 SSC-A SSC-H FSC-H Doublet discrimination Cells Doublets Singlets FSC-A SSC-W FSC-W Gate on cells Gate on doublets Gate on singlets 2n 4n Doublets >4n DAPI The BD Horizon Global Tour 24

25 Doublet discrimination Total cells Singlets Doublets Bulk sort Total cells Singlets Single-cell sort The BD Horizon Global Tour 25

26 CD14 APC CD14 APC CD14 APC Gating strategy: low antigen density populations Unstained Pre-sort Post-sort CD11b FITC CD11b FITC CD11b FITC The BD Horizon Global Tour 26

27 CD14 APC CD14 APC CD14 APC Gating strategy: low antigen density populations Unstained Pre-sort Post-sort CD11b FITC CD11b FITC CD11b FITC The BD Horizon Global Tour 27

28 CD14 APC CD14 APC CD14 APC Gating strategy: low antigen density populations Unstained Pre-sort Post-sort CD11b BV421 CD11b BV421 CD11b BV421 The BD Horizon Global Tour 28

29 Gating strategy: summary Use bivariate plots rather than histograms. Use contour plots for a clearer identification of populations of interest. Manually adjust the biexponential scale to gate all the cells of interest. Use proper controls to identify and eliminate background (autofluorescence). Use a doublet discrimination strategy for proper isolation of a single-cell suspension. The BD Horizon Global Tour 29

30 Considerations for cell sorting Panel design Fluorochrome choice, dead cell exclusion, lineage exclusion/depletion The BD Horizon Global Tour 30

31 CD14 APC CD14 APC Why is it relevant to design an optimized panel for cell sorting? Best practices to build an optimized panel for analysis apply to the cell sort as well. Additional considerations may be taken in account when designing a panel for cell sorting to obtain: Highest purity and yield Clear resolution from unwanted cell populations 18% 22% CD11b FITC CD11b BV421 The BD Horizon Global Tour 31

32 How to build a panel for cell sorting? A good panel for sorting relies on the use of negative as well as positive markers. Properly choose fluorochromes. Antigen density Spillover Co-expression Know the biology. Minimize spillover into the most critical markers to maximize the resolution of your population of interest. Exclude unwanted cells to increase the resolution of the target cells. Dead cells Lineage The BD Horizon Global Tour 32

33 Choosing fluorochromes for a cell sorting panel: exclude unwanted cells Choices for dead-cell exclusion Choices for lineage exclusion Dead cells Lineage cells Cells of interest The BD Horizon Global Tour 33

34 SSC-A Dead-cell exclusion The presence of dead cells impacts cell sorting. Inaccurate quantification of the population of interest Reduced purity Dead cells can be excluded using: Light scatter properties Viability dyes FSC-A The BD Horizon Global Tour 34

35 SSC-A SSC-A SSC-A SSC-A SSC-A SSC-A Dead-cell exclusion by light scatter Live and heat-killed HeLa cells FSC-A Viability dye FSC-A Mouse bone marrow FSC-A Viability dye FSC-A Scatter alone can be used to identify heat-killed HeLa cells. A viability dye is required to detect and gate out dead cells. The BD Horizon Global Tour 35

36 Dead-cell exclusion using viability dyes Dye Unfixed cells Fixed cells Detector Laser DAPI X BV421 UV/Violet Via-Probe Green X FITC Blue Nucleic acid dyes bind nucleic acids non-covalently PI X PE Blue/YG 7-AAD X PerCP- Cy 5.5 DRAQ7 X APC Red Via-Probe Red X APC Blue/YG Red FVS450 BV421 Violet FVS510 BV510 Violet FVS575V BV605 Violet FVS520 FITC Blue FVS570 PE Blue/YG FVS620 PE-CF594 Blue/YG No-wash stain procedure Recommended for sort of unfixed samples Fixable Viability Stains bind amine moieties covalently Wash is required after stain Recommended for sort of fixed samples FVS660 APC Red FVS700 AF700 Red FVS780 APC-H7 Red

37 Choosing fluorochromes for a cell sorting panel: exclude unwanted cells Choices for dead-cell exclusion Choices for lineage exclusion Dead cells Lineage cells Cells of interest The BD Horizon Global Tour 37

38 Lineage-cell exclusion The presence of lineage cells impacts cell sorting. Inaccurate quantification of the population of interest Reduced purity Increased time necessary to sort a rare population Lineage cells can be excluded using: Light scatter properties Lineage cocktails The BD Horizon Global Tour 38

39 SSC-A APC SSC-A APC Lineage exclusion by light scatter FSC-A CD4 FITC FSC-A CD4 FITC In peripheral blood, different cell lineages can be easily discriminated based on light scatter. The BD Horizon Global Tour 39

40 SSC-A c-kit SSC-A Lineage exclusion by light scatter is not sufficient for rare population detection FSC-A Sca-1 FSC-A In samples such as mouse bone marrow, detection of rare stem cells is confounded by the overwhelming presence of lineage cells. The use of lineage markers is needed to clearly detect rare populations of interest. The BD Horizon Global Tour 40

41 Examples of lineage marker cocktails Lineage cocktail Marker Fluorochrome T cells CD3 FITC B cells CD19 APC NK cells CD56 PerCP-Cy5.5 Monocytes/macrophages CD14 BV421 Erythrocytes CD235a BV786 Take into consideration instrument configuration and available detectors. The BD Horizon Global Tour 41

42 Examples of lineage marker cocktails Lineage cocktail Marker Fluorochrome T cells CD3 PerCP-Cy5.5 B cells CD19 PerCP-Cy5.5 NK cells CD56 PerCP-Cy5.5 Monocytes/macrophages CD14 PerCP-Cy5.5 Erythrocytes CD235a PerCP-Cy5.5 Take into consideration instrument configuration and available detectors. Combine all lineage markers in the same format to overcome configuration limitations and to increase panel design flexibility. The BD Horizon Global Tour 42

43 Choosing a fluorochrome for a lineage cocktail Fluorochrome FITC PerCP-Cy5.5 APC Alexa Fluor 700 BV421 Viability dye BD Via-Probe Green, FVS520 7-AAD, FVS620 BD Via-Probe Red, FVS660 DRAQ7, FVS700 DAPI, FVS450 Match the fluorochrome for the lineage cocktail with a viability dye detected in the same channel. In a single channel (dump channel), lineage and dead cells can now be excluded. Choose moderate dyes with high spillover into other detectors for the dump channel. Reserve dyes with bright signal and low spillover for the population of interest. The BD Horizon Global Tour 43

44 SSC-A SSC-A CD-Y Alexa Fluor 647 CD-Y Alexa Fluor 647 SSC-A SSC-A SSC-A SSC-A Building a lineage dump channel PerCP-Cy5.5 Lineage 1 PerCP-Cy5.5 Lineage 2 PerCP-Cy5.5 Lineage 3 PerCP-Cy5.5 No lineage exclusion Lineage exclusion 2.4% 15.4% 7-AAD All lineages PerCP-Cy AAD CD-X BV421 CD-X BV421 The BD Horizon Global Tour 44

45 Advantages of depleting lineage cells prior to cell sort Lineage cells can be removed from the sample prior to sort using multiple rounds of magnetic selection or cell sorting Cell enrichment Cell depletion Lineage depletion can improve cell sorts of rare population of cells Increased sort efficiency Increased purity Reduced sort time The BD Horizon Global Tour 45

46 HLA-DR V450 CD123 PE HLA-DR V450 CD123 PE Magnetic depletion of lineage cells reduces sorting time No lineage depletion 0.28% pdcs 0.075% Theoretical time to sort 10 5 pdcs 6.5 hours Lineage FITC 1 st round of lineage depletion 21.6% CD11c APC pdcs 10.7% Theoretical time to sort 10 5 pdcs 2.5 minutes 46 Lineage FITC CD11c APC The BD Horizon Global Tour 46

47 CD123 PE CD123 PE SSC-A HLA-DR V450 SSC-A HLA-DR V450 and increases purity No lineage depletion 1 st round magnetic lineage depletion FSC-A lin FITC FSC-A lin FITC CD11c APC CD11c APC The BD Horizon Global Tour 47

48 Choosing fluorochromes for a cell sorting panel: resolve the population of interest Choices for dead-cell exclusion Choices for lineage exclusion Choices for populations of interest Dead cells Lineage cells Cells of interest The BD Horizon Global Tour 48

49 c-kit Detection of murine hematopoietic stem and progenitor cells Myeloid Bone marrow GMLP HSC Hematopoietic stem cells (HSCs) and common lymphoid progenitors (CLPs) are rare cell populations (<1%) in mouse bone marrow. CD127 + CLP CLP2 HSCs: bright expression of c-kit and Sca-1 CLPs: dim expression of c-kit and Sca-1 pro-b Sca-1 B cell Clear resolution of dim and bright c-kit and Sca-1 populations is critical for the isolation of HSCs and CLPs. Adapted from Iwasaki H, Akashi K. Hematopoietic developmental pathways: on cellular basis. Oncogene. 2007; 26: The BD Horizon Global Tour 49

50 Impact of fluorochrome choice on HSC and CLP resolution Antigen Specificity Lineage/7-AAD Sca-1 c-kit CD127 The BD Horizon Global Tour 50 Assignment Fluorochrome BV421 BV650 PE PE-CF594 BB515 APC/Alexa Fluor 647 BV605 BV786 BV510 FITC/Alexa Fluor 488 PerCP-Cy5.5 V450 V500 Alexa Fluor 700 APC-H7 Laser

51 c-kit APC-H7 Count Fluorochrome choice: panel 1 HSCs CLPs Sca-1 PE CD127 APC Antigen density: APC-H7 is not bright enough to clearly separate dim CLPs. Adjacent spillover: Co-expression of c-kit and CD127 was not taken into consideration. The BD Horizon Global Tour 51

52 Impact of fluorochrome choice on HSC and CLP resolution Antigen Specificity Assignment Fluorochrome Laser Lineage/ 7-AAD Sca-1 c-kit CD127 BV421 BV650 PE PE-CF594 BB515 APC/Alexa Fluor 647 BV605 BV786 BV510 The BD Horizon Global Tour 52 FITC/Alexa Fluor 488 PerCP-Cy5.5 V450 V500 Alexa Fluor 700 APC-H7

53 c-kit BV421 Count Fluorochrome choice: panel 2 HSCs CLPs Sca-1 PE CD127 APC Antigen density: BV421 clearly separates dim and bright c-kit positive cells. Adjacent spillover: Fluorochromes were spread across different lasers, for minimal spectral overlap, maximum resolution. The BD Horizon Global Tour 53

54 c-kit APC-H7 c-kit BV421 Fluorochrome choice: panel comparison Sca-1 PE Sca-1 PE The BD Horizon Global Tour 54

55 Recipe for best panel for sorting Use a dump channel to exclude dead and lineage cells. Magnetic depletion of lineage cells further improves the cell sort of rare populations. Take into consideration co-expression and spillover. Match the brightest fluorochromes with the antigens with lower antigen density. The BD Horizon Global Tour 55

56 Conclusion Choices for dead-cell exclusion Choices for lineage exclusion Choices for populations of interest Dead cells Lineage cells Cells of interest The BD Horizon Global Tour 56