A step-by-step approach to build and analyze a multicolor panel
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1 Analyze A step-by-step approach to build and analyze a multicolor panel 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. The 2016 BD BD. Horizon BD, the BD Global Logo and Tour all other 1trademarks are property of Becton, Dickinson and Company.
2 Best practices for building and analyzing panels CD3 + Reagents Controls FMO CD4 + CD4 -?? Resolution CD25 + CD127 + Impact of spread on resolution Multistep analysis approach Build and analyze panels Activity The BD Horizon Global Tour 2
3 CD56 CD56 Fluorescence spillover and spread impact resolution CD3 CD3 Understanding the impact of fluorescence spillover and spread is the key to good panel design. The BD Horizon Global Tour 3
4 Spread impacts resolution Spillover introduces spread and background The BD Horizon Global Tour 4
5 PE Spillover introduces background and spread SOV = 9.15% PE-Cy 5 The amount of the spread Amount of spillover The BD Horizon Global Tour 5
6 PE Spillover introduces background and spread SOV = 0.86% FITC The amount of the spread Amount of spillover The BD Horizon Global Tour 6
7 PE Spillover introduces background and spread SOV = 26.40% PE-CF594 The amount of the spread Amount of spillover The BD Horizon Global Tour 7
8 PE Spillover introduces background and spread MFI = 35,000 7,000 1, PE-CF594 The amount of the spread Amount of spillover Reagent brightness The BD Horizon Global Tour 8
9 PE Spillover introduces background and spread Population resolution for the PE-CF594 fluorescence parameter is decreased by increased spread due to PE spillover from another fluorescence parameter. To maximize the resolution of a given double-positive subpopulation: Minimize fluorescence spread into the detector that defines that population PE-CF594 The amount of the spread Amount of spillover ( Antigen Fluorochrome ) density brightness The BD Horizon Global Tour 9
10 PE PerCP-Cy5.5 Quantifying the impact of fluorescence spillover 50 Volts Ref Voltage 165% 44% Spillover Values (SOVs) Are totally dependent upon gain settings (PMTVs). 18.1% 15.4% Does not always accurately reflect the impact of spread. CD4 FITC CD4 PE The BD Horizon Global Tour 10
11 BD FACSCelesta flow cytometer B/V/UV BUV737 spread ~400 ~480 ~530 ~570 ~610 ~660 ~720 ~780 UV BUV395 BUV395 BUV737 BV510 BV421 BV510 BV605 BV650 BV711 BV786 Violet BB515 PE PE-CF594 PerCP-Cy5.5 Blue CD4 BUV737
12 PerCP-Cy5.5 PE-CF594 BV605 BUV737 Spread impacts the resolution of double-positive populations CD4 BV650 CD4 BV650 Spread is most important when considering reagents for antigens co-expressed on a subpopulation. Double-positive populations need to be resolved from the single-positive populations. Although the spread of BV650 is equivalent into BV605 & BUV737, it has less of an impact on the resolution of a marker stained with BUV737. Although the spread of PE is equivalent into PerCP-Cy 5.5 and PE-CF594, it has less of an impact on the resolution of a marker stained with PE-CF594. CD4 PE CD4 PE
13 CD A (PE-CF594) Resolution of double-positive populations SI 700 A + Loss of Resolution CD B (PE) DP-SI 50 A + B + A - A - B + Here is a classic resolution of a single-positive (A + ) population being resolved from a negative (A - ) population. Resolution measured in Stain Index (SI) Adding a second co-expressed marker, we now have to resolve a double-positive (A + B + ) population from a single (A - B + ) population. Resolution measured in a doublepositive Stain Index (DP-SI) The spread of fluorochrome B into the A detector reduces the resolution of a double-positive (A + B + ) population from the A - B + population. The BD Horizon Global Tour 13
14 Double-positive stain index matrix Double-Positive Stain Index* Co-expressed Fluor Primary Fluor FITC PE PerCP- Cy5.5 PE-Cy7 APC APC- H7 FITC PE PerCP-Cy PE-Cy APC APC-H This table shows double-positive Stain Index values A double-positive Stain Index less than the single-positive Stain Index indicates that the spread of the secondary coexpressed fluorochrome has reduced resolution. * Run on a BD FACSVerse flow cytometer The BD Horizon Global Tour 14
15 Double-positive stain index matrix Double-Positive Stain Index* Co-expressed Fluor Primary Fluor FITC PE PerCP- Cy5.5 PE-Cy7 APC APC- H7 FITC PE PerCP-Cy PE-Cy APC APC-H * Run on a BD FACSVerse This table shows double-positive Stain Index values A double-positive Stain Index less than the single-positive Stain Index indicates that the spread of the secondary coexpressed fluorochrome has reduced resolution. This table shows the relative loss of resolution which can be evaluated as the percent difference between the Stain Index and the double-positive Stain Index 1- (50/700) x 100%. The BD Horizon Global Tour 15
16 The resolution impact matrix Resolution Impact Matrix* Co-expressed Fluor Primary Fluor FITC PE FITC 68 PE 340 PerCP-Cy PerCP- Cy5.5 PE-Cy7 PE-Cy7 522 APC APC- H7 APC 486 APC-H7 116 * Run on a BD FACSVerse % loss of SP-SI <20% 20-40% 40-60% 60-80% >80% SI (Primary) This table shows double-positive Stain Index values A double-positive Stain Index less than the single-positive Stain Index indicates that the spread of the secondary coexpressed fluorochrome has reduced resolution. This table shows the relative loss of resolution which can be evaluated as the percent difference between the Stain Index and the double-positive Stain Index 1- (50/700) x 100%. Color coding the percent loss makes it easy to visualize which fluorochromes have the greatest impact on each other. The BD Horizon Global Tour 16
17 Using the resolution impact matrix The resolution impact matrix provides a quick visual tool to help assess potential problems with spread when evaluating potential use of two fluorochromes for co-expressed markers on a population of cells. Primary Fluor FITC PE FITC 68 PE 340 PerCP-Cy * Run on a BD FACSVerse Resolution Impact Matrix* Co-expressed Fluor PerCP- Cy5.5 PE-Cy7 PE-Cy7 522 APC APC- H7 APC 486 APC-H7 116 % loss of SP-SI <20% 20-40% 40-60% 60-80% >80% SI (Primary) The table shows: Adding a PE reagent to a coexpressed marker will have significant spread into and a major negative impact on the resolution of the double-positive cells in the PerCP-Cy5.5 detector. No fluorochromes have a significant negative impact on the resolution of FITC + cells. FITC has minimal impact on any other fluorochrome. The BD Horizon Global Tour 17
18 Best practices for building and analyzing panels CD3 + Reagents Controls FMO CD4 + CD4 -?? Resolution CD25 + CD127 + Impact of spread on resolution Multistep analysis approach Build and analyze panels Activity The BD Horizon Global Tour 18
19 Multistep analysis approach A systematic process for the correct analysis of data The BD Horizon Global Tour 19
20 The panel design process MARKER A MARKER B A+ B+ No problem with spillover/spread NO Assign fluorochromes based upon antigen density. Are they co-expressed? A>>B YES A+ B+ Relative Expression B>>A Reiterate Your Panel Choose Reagents For B use a bright fluorochrome Test Your Panel For A use a dim fluorochrome that causes minimal loss of resolution of the A + B - population Understand- Why? NO Do you get good resolution? YES SUCCESS
21 Goal To assess if data from a flow cytometry assay is optimal What does optimal mean? Can I resolve each of the critical populations in the panel? Can I easily draw gates for each of the populations identified with this assay? If answers to both of these questions are YES, then panel can be considered optimal. If answers to either of the questions is NO, then you should try another iteration of the panel design. The BD Horizon Global Tour 21
22 Step-by-step analysis 1. Assess adequacy of instrument performance and setup Controls used CST Reports / L-J Plots Unstained 2. Assess reagent performance Single Stain Assess compensation accuracy Assess protocol impacts (when necessary) Single Stain / Comp Biological Assess level of resolution for each marker Identify any sources of loss resolution Single Stain FMO The BD Horizon Global Tour 22
23 Step 1: assess adequacy of instrument performance and setup V450 Qr PE Qr V450 Detector Voltage The BD Horizon Global Tour 23
24 Relative Cell Number Count Step 2: assess reagent performance Reference TDS Experimental TDS shows surface staining. Experimental is IC protocol. APC-H7 CD3, with isotype control CD3 APC-H7 Conclusion: need to improve staining conditions. The BD Horizon Global Tour 24
25 CD127 BV421 Step 3: assess compensation accuracy Single-stained control BV711 into BV421 appears to be over compensated. Population center is below zero Population is on a diagonal CD45RA BV711 Both are indications that there is an error in the compensation. The BD Horizon Global Tour 25
26 Step 4: assess protocol impacts Single stain control vs Biological control Impact of culturing / activation on antigen resolution PBMCs (Fresh) PBMCs stimulated Antigen resolution is reduced because: CD4 PE-Cy7 Stimulation increased the background staining and reduced antigen expression. Impact of intracellular staining CD8 APC-H7 Surface stain Intracellular stain Intracellular staining increased the background staining. Impact of fixation on surface staining CD127 BV421 Stain (no fixation) Stain (PermBuffer III) The fixation reduced the fluorescence of the BV421. The BD Horizon Global Tour 26
27 Step 5: assess level of resolution for each marker Here there is little or no difference in the profiles of the single stained reagents and the multicolor tube. Marker resolution is not affected by the panel design. Singlestained SI: 72 SI: 129 SI: 201 SI: 32 SI: 75 SI: 65 SI: 127 SI: 200 SI: 33 SI: 84 Panel CD4 BUV395 CD3 BV421 CD8 FITC CD27 PE-CF594 CD45RA APC The BD Horizon Global Tour 27
28 Step 5: assess level of resolution for each marker Single-stained control Panel IFNg AF700 Here, there is a clear loss of resolution of the dim IFNg (Alexa Fluor 700) cells. The BD Horizon Global Tour 28
29 Step 6:identify sources of resolution loss Single-stained control Panel FMO BV711 IFNg AF700 Here, there is a clear loss of resolution of the dim IFNg (Alexa Fluor 700) cells. The BV711 FMO control confirms that the loss of resolution in the Alexa Fluor 700 detector is due to the BV711 reagent. The BD Horizon Global Tour 26
30 Review - experimental controls Controls are used to identify and resolve issues when optimizing a new multicolor panel Unstained controls to highlight the background or autofluorescence of the biological system to optimize instrument setup Single stained controls to QC the compensation to assess any impact of marker resolution in the panel Fluorescence Minus One (FMO) controls to help identify potential impact of spillover/spread impacting resolution in the panel
31 Best practices for building and analyzing panels CD3 + Reagents Controls FMO CD4 + CD4 -?? Resolution CD25 + CD127 + Impact of spread on resolution Multistep analysis approach Build and analyze panels Activity The BD Horizon Global Tour 31
32 Build and analyze panels Apply the panel design rules to achieve the best resolution for your population of interest The BD Horizon Global Tour 32
33 Building a 4-color panel to identify regulatory T-cells (Tregs) Experimental goal: Identify Treg cells Design the best panels for different instrument configurations Markers used: CD3, CD4, CD25, CD127 Gating strategy CD3 + CD4 + CD4 - CD25 + CD127 + The BD Horizon Global Tour 33
34 No. PE molecules Grouping antigen density: T-cells Average number of molecules on T cells 100,000 Ag density Classification 40,000 10,000 CD3 High Primary CD4 High Primary CD127 Medium Secondary CD25 Low Tertiary 4,000 1, The BD Horizon Global Tour 34
35 Approaches to panel design on BD Accuri C6 Plus Laser Blue (488 nm) Red (640 nm) Fluorochrome FITC BB515 PE PerCP-Cy 5.5 APC 3 detectors off the blue and 1 off the red laser: possible spillover issues. The BD Horizon Brilliant dye BB515 offers an additional choice for a bright fluorochrome. The BD Horizon Global Tour 35
36 BD Accuri C6 Plus panel 1 Antigen Specificity CD4 CD3 CD127 CD25 Assignment Fluorochrome PE APC/Alexa Fluor 647 FITC/Alexa Fluor 488 PerCP-Cy5.5 Laser The BD Horizon Global Tour 36
37 CD3 PerCP-Cy5.5 CD127 AF647 Count BD Accuri C6 Plus panel 1 CD4 PE CD25 FITC CD25 FITC
38 BD Accuri C6 Plus panel 2 Antigen Specificity CD4 CD3 CD127 CD25 Assignment Fluorochrome PE BB515 APC/Alexa Fluor 647 PerCP-Cy5.5 Laser The BD Horizon Global Tour 38
39 CD3 PerCP-Cy5.5 CD127 AF647 Count BD Accuri C6 Plus panel 2 CD4 PE CD25 BB515 CD25 BB515
40 Approaches to panel design on BD FACSCelesta Blue/Violet (B/V) Laser Blue (488 nm) Violet (405 nm) Fluorochrome FITC BB515 PE PE-CF594 PerCP-Cy5.5 V450 V500 Four detectors off the blue and five off the violet laser: reduced spillover issues. Before the BD Horizon Brilliant dyes, only two dim violet dyes were available. The BD Horizon Global Tour 40
41 Approaches to panel design on BD FACSCelesta Blue/Violet (B/V) Laser Blue (488 nm) Violet (405 nm) Fluorochrome FITC BB515 PE PE-CF594 PerCP-Cy5.5 BV421 BV480 BV510 BV605 BV650 BV711 BV786 Four detectors off the blue and five off the violet laser: reduced spillover issues. Before the BD Horizon Brilliant dyes, only two dim violet dyes were available. Seven moderate/bright BD Horizon Brilliant dyes are now available. The BD Horizon Global Tour 41
42 Building a panel on BD FACSCelesta B/V panel 1 Antigen Specificity CD4 CD3 CD127 CD25 Assignment Fluorochrome PE PE-CF594 PerCP-Cy5.5 FITC/Alexa Fluor 488 V450 Laser V500 The BD Horizon Global Tour 42
43 CD3 V500 CD127 FITC BD FACSCelesta B/V panel 1 CD4 V450 CD25 PE The BD Horizon Global Tour 43
44 Building a panel on BD FACSCelesta B/V - panel 2 Antigen Specificity CD4 CD3 CD127 CD25 Assignment Fluorochrome BV421 BV650 BV711 PE PE-CF594 BB515 BV786 BV605 BV510 FITC PerCP-Cy5.5 V450 V500 Laser The BD Horizon Global Tour 44
45 CD3 PE-CF594 CD127 BV786 BD FACSCelesta B/V panel 2 CD4 FITC CD25 BV421 The BD Horizon Global Tour 45
46 Building a 6-color panel to identify T-cell subsets Experimental goal: Drop in 2 markers (CD45RA and CD197) to identify Tregs and memory/effector T-cells Markers used: CD3, CD4, CD25, CD127, CD45RA, CD197 Assign antigen expression levels Gating strategy CD3 + CD4- CD4 + Ag density Classification CD3 High Primary CD4 High Primary CD25 + CD127 +/- CD45RA +/- CD45RA +/- CD197 +/- CD197 +/- CD45RA High Secondary CD127 Medium Secondary CD197 Medium Secondary CD25 Low Tertiary The BD Horizon Global Tour 46
47 Approaches to panel design on BD FACSCelesta Blue/Violet/Red (BVR) Laser Blue (488 nm) Violet (405 nm) Red (640nm) Fluorochrome FITC BB515 PE PE-CF594 PerCP-Cy 5.5 BV421 BV480 BV510 BV605 BV650 BV786 APC APC-R700 APC-H7 The addition of the red laser allows fluorochromes to be spread across 3 lasers. Minimize spillover and maximize resolution. The BD Horizon Global Tour 47
48 Building a 6-color panel on BD FACSCelesta B/V/R Antigen Specificity CD4 CD3 CD127 CD45RA CD197 CD25 Assignment Fluorochrome BV421 BV650 PE PE-CF594 BB515 APC/Alexa Fluor 647 BV786 BV605 BV510 FITC PerCP-Cy5.5 V450 V500 Alexa Fluor 700 APC-H7 Laser The BD Horizon Global Tour 48
49 CD45RA APC CD45RA APC CD3 APC-H7 CD127 BV786 6-color panel on BD FACSCelesta B/V/R CD4 FITC CD25 BV421 CD197 PE-CF594 CD197 PE-CF594 The BD Horizon Global Tour 49
50 Approaches to panel design on BD FACSCelesta B/V/UV configuration Laser Blue (488 nm) Violet (405 nm) UV (355nm) Fluorochrome FITC BB515 PE PE-CF594 PerCP-Cy 5.5 BV421 BV480 BV510 BV605 BV650 BV711 BV786 BVUV395 BVUV737 The addition of the ultraviolet laser allows fluorochromes to be spread across 3 lasers. Minimize spillover and maximize resolution. The use of BUV395 facilitates panel design Lack of spillover in any other channel. Not impacted by the majority of the other dyes. The BD Horizon Global Tour 50
51 BD FACSCelesta B/V/UV BUV395 spread ~480 ~530 ~570 ~ 610 ~660 ~720 ~780 BV421 BV510 BV605 BV650 BV711 BV786 Violet Laser Blue Laser FITC PE PE-CF594 PerCP-Cy5.5 BUV737 UV Laser CD4 BUV395 The BD Horizon Global Tour 51
52 Designing a 6-color panel on BD FACSCelesta B/V/UV Antigen Specificity CD4 CD3 CD127 CD45RA CD197 CD25 The BD Horizon Global Tour 52 Assignment Fluorochrome BV421 BV650 BV711 PE PE-CF594 BB515 BV786 BUV737 BV605 BUV395 BV510 FITC PerCP-Cy5.5 V450 V500 Laser
53 CD45RA BUV395 CD45RA BUV395 CD3 BV510 CD127 BV786 6-color panel on BD FACSCelesta B/V/UV CD4 PerCP-Cy5.5 CD25 BB515 CD197 BV421 CD197 BV421 The BD Horizon Global Tour 53
54 CD127 FITC CD127 BV786 CD127 BV786 Summary FACSCelesta B/V FACSCelesta B/V/R FACSCelesta B/V/UV CD25 PE CD25 BV421-A CD25 BB515 The BD Horizon Global Tour 54
55 Minimal spectral overlap panels Strategies to maximize resolution by minimizing spillover and compensation The BD Horizon Global Tour 55
56 Multiple lasers for multicolor analysis Red (640 nm) Blue (488 nm) Three lasers allow for the simultaneous analysis of multiple fluorescent parameters. Violet (405 nm)
57 Multiple lasers for minimal spectral overlap panels Red (640 nm) APC One color off each laser Blue (488 nm) FITC Take into consideration: - Residual spillover - Cross-laser excitation - Antigen density Violet (405 nm) BV421
58 Designing a 3-color minimal spectral overlap panel on BD FACSCelesta BVR Antigen Specificity CD4 CD127 CD25 Assignment Fluorochrome BV421 BV650 PE PE-CF594 BB515 APC/Alexa Fluor 647 BV786 BV605 BV510 FITC/Alexa Fluor 488 PerCP-Cy5.5 V450 V500 Alexa Fluor 700 APC-Cy7 Laser The BD Horizon Global Tour 58
59 SSC-A SSC-A CD127 AF647 SSC-A SSC-A CD127 AF647 3-color minimal compensation panel on BD FACSCelesta B/V/R Compensated Compensation matrix Fluor -% Fluor Spectral Overlap 6.9% FITC 0.29 BV421 AF FSC-A CD4 FITC Not compensated CD25 BV421 BV FITC AF BV AF647 FITC % FSC-A CD4 FITC CD25 BV421
60 Multiple lasers for minimal spectral overlap panels UV (355 nm) BUV395 One color off each laser Blue (488 nm) FITC PerCP-Cy5.5 Two colors off one laser if the spectra are well separated Violet (405 nm) BV421 Take into consideration: - Residual spillover - Cross-laser excitation - Antigen density
61 4-color minimal compensation panel on BD FACSCelesta B/V/UV Antigen Specificity CD4 CD3 CD127 CD25 Assignment Fluorochrome BV421 BV650 BV711 PE PE-CF594 BB515 BV786 BUV737 BV605 BUV395 BV510 FITC/Alexa Fluor 488 PerCP-Cy5.5 V450 V500 Laser The BD Horizon Global Tour 61
62 CD3 BUV395 CD127 BV421 CD3 BUV395 CD127 BV421 4-color minimal spectral overlap panel on BD FACSCelesta B/V/UV Compensated 8.4% CD4 PerCP-Cy5.5 CD25 BB515 Not compensated 8.4% Compensation matrix BB515 Fluor -% Fluor Spectral Overlap 0.08 BUV395 BV PerCP-Cy BV BUV395 BB PerCP-Cy BB515 PerCP BV421 Cy BUV BB BV421 BUV PerCP-Cy CD4 PerCP-Cy5.5 CD25 BB515
63 5-color minimal compensation panel on BD LSRFortessa X20 cell analyzer Antigen Specificity CD4 CD3 CD127 CD45RA CD25 The BD Horizon Global Tour 63 Assignment Fluorochrome BV421 BV650 BV711 PE PE-CF594 PE-Cy7 BB515 APC/Alexa Fluor 647 BV786 BUV737 BV605 BUV395 BV510 FITC/Alexa Fluor 488 PerCP-Cy5.5 Alexa Fluor 700 APC-Cy7 Laser
64 CD3 FITC CD127 AF647 CD45RA PE-CF594 CD3 FITC CD127 AF647 CD45RA PE-CF594 5-color minimal spectral overlap panel on BD LSRFortessa X20 cell analyzer Compensated Compensation matrix Fluor -% Fluor Spectral Overlap 8.4% BV BUV FITC AF PE-CF FITC 0.00 CD4 BUV395 CD25 BV421 CD25 BV421 BUV395 AF647 BV Not compensated 8.2% PE-CF FITC 0.01 BV421 BUV AF PE-CF FITC 0.12 BV AF647 BUV PE-CF CD4 BUV395 CD25 BV421 CD25 BV421 FITC 0.18 BV BUV395 PE-CF AF
65 Best practices for building and analyzing panels CD3 + Reagents Controls FMO CD4 + CD4 -?? Resolution CD25 + CD127 + Impact of spread on resolution Multistep analysis approach Build and analyze panels Activity The BD Horizon Global Tour 65
66 Activity Complete a 6-color panel for the optimal resolution of Tregs The BD Horizon Global Tour 66
67 Activity Gating strategy Marker Antigen Density Dye CD3 High BV510 CD3 + CD4 High PerCP-Cy5.5 CD25 Low? CD127 Medium? CD197 Medium BV421 CD4 + CD4- CD45RA High BUV395 CD25 + CD127 +/- CD45RA +/- CD45RA +/- CD197 +/- CD197 +/- Complete the panel by assigning fluorochromes for a good resolution of CD25 high CD127 low Tregs. Take into consideration: Co-expression Antigen density Spillover The BD Horizon Global Tour 67
68 Panel 1 Antigen Specificity Assignment Fluorochrome Laser CD197 BV421 CD127 CD25 BV650 BV711 PE PE-CF594 BB515 BV786 BUV737 BV605 CD45RA CD3 BUV395 BV510 FITC/Alexa Fluor 488 CD4 PerCP-Cy5.5 V450 V500 The BD Horizon Global Tour 68
69 CD45RA BUV395 CD45RA BUV395 CD3 BV510 CD127 BUV737 Panel 1 review antigen density FITC CD4 PerCP-Cy5.5 BB515 CD25 FITC BV421 CD197 BV421 CD197 BV421 CD25
70 Panel 2 Antigen Specificity CD127 CD25 CD197 CD45RA CD3 CD4 Assignment Fluorochrome BV421 BV650 BV711 PE PE-CF594 BB515 BV786 BUV737 BV605 BUV395 BV510 FITC/Alexa Fluor 488 PerCP-Cy5.5 V450 V500 Laser The BD Horizon Global Tour 70
71 CD45RA BUV395 CD45RA BUV395 CD3 BV510 CD127 PE Panel 2 adjacent spillover CD4 PerCP-Cy5.5 CD25 BV605 CD197 BV421 CD197 BV421
72 Full panel review Single stain CD45RA BUV395 CD197 BV421 CD3 BV510 CD25 BV605 CD127 PE CD4 PerCP-Cy5.5 Panel CD45RA BUV395 CD197 BV421 CD3 BV510 CD25 BV605 CD127 PE CD4 PerCP-Cy5.5 The BD Horizon Global Tour 72
73 Identify sources of resolution loss Single stain Panel CD25 BV605 CD25 BV605 FMO CD4 BUV395 FMO CD197 BV421 FMO CD3 BV510 FMO CD127 PE FMO CD4 PerCP-Cy5.5 CD25 BV605 The BD Horizon Global Tour 69
74 Panel 3 Antigen Specificity CD127 CD25 CD197 CD45RA CD3 CD4 Assignment Fluorochrome BV421 BV650 BV711 PE PE-CF594 BB515 BV786 BUV737 BV605 BUV395 BV510 FITC / Alexa Fluor 488 PerCP-Cy5.5 V450 V500 Laser The BD Horizon Global Tour 74
75 CD45RA BUV395 CD45RA BUV395 CD3 BV510 CD127 PE Panel 3 review residual spillover Single stain CD4 PerCP-Cy5.5 CD25 PE-CF594 Panel FMO PE CD197 BV421 CD197 BV421 CD25 PE-CF594
76 Panel 4 Antigen Specificity CD127 CD25 CD197 CD45RA CD3 CD4 Assignment Fluorochrome BV421 BV650 BV711 PE PE-CF594 BB515 BV786 BUV737 BV605 BUV395 BV510 FITC/Alexa Fluor 488 PerCP-Cy5.5 V450 V500 Laser The BD Horizon Global Tour 76
77 CD45RA BUV395 CD45RA BUV395 CD3 BV510 CD127 BV786 Panel 4 good choice! Single stain CD4 PerCP-Cy5.5 CD25 BB515 Panel CD25 BB515 CD197 BV421 CD197 BV421
78 Full panel review Single stain CD3 BV510 CD4 PerCP-Cy5.5 CD127 BV786 CD25 BB515 CD45RA BUV395 CD197 BV421 Panel CD3 BV510 CD4 PerCP-Cy5.5 CD127 BV786 CD25 BB515 CD45RA BUV395 CD197 BV421 The BD Horizon Global Tour 78
79 CD127 BUV737 CD127 PE CD127 PE CD127 BV786 Activity summary Panel 1 Panel 2 Panel 3 Panel 4 CD25 FITC CD25 BV605 CD25 PE-CF594 CD25 BB515 The BD Horizon Global Tour 79
80 Conclusion CD3 + Reagents Controls FMO CD4 + CD4 -?? Resolution CD25 + CD127 + Impact of spread on resolution Multistep analysis approach Build and analyze panels Activity The BD Horizon Global Tour 80
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