U SE THE SSM AND GATING TREE TO GUIDE ANTIBODY - FLUOROCHROME - - PowerPoint PPT Presentation

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U SE THE SSM AND GATING TREE TO GUIDE ANTIBODY - FLUOROCHROME - - PowerPoint PPT Presentation

Jul 31, 2023 425 likes •587 views

A NTIGEN C ATEGORIZATION Marker expression usually comes in three different patterns: On/Off expression, usually lineage markers (CD3, CD4, CD19) Intermediate or continuous expression patterns (CD45RA, CD38, CD57, cytokines, many more)

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SLIDE 1

ANTIGEN CATEGORIZATION

  • Marker expression usually comes in three different patterns:
  • On/Off expression, usually lineage markers (CD3, CD4, CD19)
  • Intermediate or continuous expression patterns (CD45RA,

CD38, CD57, cytokines, many more)

  • Dimly expressed or rare markers

ØChoose detectors that receive low spillover and pair with bright fluorochromes

Image courtesy of Florian Mair

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SLIDE 2

USE A GATING TREE TO ASSESS CO-EXPRESSION OF MARKERS

Live CD14+ CD14- CD3- CD3+ CD8+ CD4+ FOXP3+ CD19+

These cells will co-express FOXP3, CD4, and CD3 These cells express CD19, but nothing else These cells express CD14, but nothing else CD4 and CD8 T cells will co-express CD3, but they are mutually exclusive for CD4 and CD8 PBMC

FOXP3-

These cells will co-express CD4 and CD3, but not FOXP3

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SLIDE 3

ASSESS COMMERCIAL AVAILABILITY OF CONJUGATED FLUOROCHROMES

  • Fluorofinder – online resource/database that shows all commercially available

antibody-fluorochrome conjugates

Øwww.fluorofinder.com ØAlso has an online panel builder and spectra viewer

  • Use filter to narrow results for

ØTarget species ØCompany (or you will get false positives from Biorbyt) ØFluorochrome (if you want) ØClone (if you want)

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SLIDE 4

USING FLUORFINDER

Click on “Search Antibodies” Enter antigen of interest Narrow down results using the filters Scroll through fluorochromes to check availability

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SLIDE 5

USE THE SSM AND GATING TREE TO GUIDE ANTIBODY-

FLUOROCHROME PAIRINGS

ØAssign bright markers (or highly/broadly expressed antigens) to channels that contribute little spillover ØAssign critical or dimly expressing makers to channels that accept little spillover ØPlace mutually exclusive combinations on channels with high spillover/spread values ØUse the spillover/spread matrix and gating tree to guide placement of co-expressing markers

B515 B610 B660 B710 B780 G575 G610 G660 G710 G780 B515 0.668 0.638 0.763 0.319 0.237 0.236 0.649 0.0928 B610 0.251 4.66 5.11 1.41 1.35 3.71 2.08 4.76 0.659 B660 0.918 2.02 7.09 1.98 2.59 1.04 3.25 6.1 0.977 B710 0.848 0.677 2.73 4.34 2.05 0.205 1.53 10.1 3.62 B780 0.713 0.538 0.637 1.34 0.537 0.335 0.342 1.22 2.38 G575 0.203 4.1 2.3 2.87 0.676 2.1 2.01 2.95 0.55 G610 0.162 5.08 4.33 6.28 1.54 2.17 3.71 6.55 1.39 G660 0.439 4.38 6.35 2.35 1.95 0.506 7.69 2.06 G710 0.362 1.38 3.54 17.9 5.04 5.27 0.953 4.41 6.3 G780 0.304 0.383 0.598 6.64 0.476 0.331 0.43 0.824 R660 0.218 1.19 1.49 0.62 0.622 0.376 1.1 1.76 0.561 R710 0.465 1.74 0.844 0.511 0.14 2.07 0.884

Ex: CD4 and CD8 on G710 and B710 are still distinguishable as both markers are mutually exclusive

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SLIDE 6

ANTIBODY TITRATION(1)

  • ALWAYS TITRATE!!!!

ØEvery clone will behave differently ØManufacturers vial at different concentrations

  • Titrate under the conditions in which the antibody will be used in the full panel

Øi.e. surface antibodies that are part of an intracellular assay must be fixed/permed

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SLIDE 7

ANTIBODY TITRATION(2)

  • Titrating will identify the optimal concentration at which to use the antibody

ØIt will (almost always) save reagent (money)

10-2 10-1 100 102 103 104 105 Titer <U780-A>: CD4

U780 CD4 BUV805 0.5

10-2 10-1 100 102 103 104 105 Titer <V510-A>: TCRgd

CD3+ V510 TCRgd BV480 2.5

10-2 10-1 100 102 103 104 105 Titer <B780-A>: CD56

B780 CD56 BB790 0.05

1/100th recommended titer 1/10th recommended titer 1/2 recommended titer

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SLIDE 8

ANTIBODY TITRATION(3)

  • Spreading error can be reduced if a saturating concentration is not needed (lineage

markers)

ØSpreading is proportional to signal intensity

Limit of detection

High titer of CD8-BB660 Lower titer of CD8-BB660

Limit of detection

Image courtesy of Florian Mair

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SLIDE 9

STAINING CONTROLS

  • Necessary to identify cells which do
  • r do not express a given antigen
  • Threshold for positivity may depend
  • n the amount of fluorescence in
  • ther channels
  • Unstained cells or isotype controls

stains are improper controls

Slide provided by M. Roederer, NIH

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SLIDE 10

FMO CONTROLS

  • FMO = Fluorescence

Minus One

Ø Cells are stained with all reagents EXCEPT the

  • ne of interest
  • Essential for complex

panels

  • Reveal unnoticed or

unexpected issues with spreading

  • Should be used for setting

correct gates

100 101 102 103 104 100 101 102 103 104 105 100 101 102 103 104 100 101 102 103 104

Unstained Control FMO Control Fully Stained PE FITC FITC PE Cy5PE Cy7PE – – – – CD3 – CD8 CD45RO CD3 CD4 CD8 CD45RO Isotype Bounds FMO Bounds

Perfetto et al, Nat Rev Immunol 2004

PBMC stained as shown. Compensation properly set.

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SLIDE 11

FMO EXAMPLE – MISSING PE-TR

FSC-A <PE Tx RD-A>: CD45RO FSC-H <PE Tx RD-A>: CD45RO SSC-A <PE Tx RD-A>: CD45RO <FITC-A>: CD103 <PE Tx RD-A>: CD45RO <QDot 655-A>: CD3 <PE Tx RD-A>: CD45RO <QDot 605-A>: CD27 <PE Tx RD-A>: CD45RO <QDot 585-A>: CD45RA <PE Tx RD-A>: CD45RO <Pacific Blue-A>: LD <PE Tx RD-A>: CD45RO <APC Cy7-A>: CD4 <PE Tx RD-A>: CD45RO <Alexa 680-A>: CD57 <PE Tx RD-A>: CD45RO <APC-A>: CD95 <PE Tx RD-A>: CD45RO <PE Cy7-A>: CCR7 <PE Tx RD-A>: CD45RO <PE Cy55-A>: CD8 <PE Tx RD-A>: CD45RO <PE Cy5-A>: CD28 <PE Tx RD-A>: CD45RO <PE Green laser-A>: CD127 <PE Tx RD-A>: CD45RO

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SLIDE 12

FMO EXAMPLE – MISSING PE-CY5

  • CD28 PE-Cy5 was

not added to the staining cocktail but there appears to be a positive signal

  • Staining artefact

comes from large spreading of Qdot reagent

FSC-A <PE Cy5-A>: CD28 FSC-H <PE Cy5-A>: CD28 SSC-A <PE Cy5-A>: CD28 <FITC-A>: CD103 <PE Cy5-A>: CD28 <QDot 655-A>: CD3 <PE Cy5-A>: CD28 <QDot 605-A>: CD27 <PE Cy5-A>: CD28 <QDot 585-A>: CD45RA <PE Cy5-A>: CD28 <Pacific Blue-A>: LD <PE Cy5-A>: CD28 <APC Cy7-A>: CD4 <PE Cy5-A>: CD28 <Alexa 680-A>: CD57 <PE Cy5-A>: CD28 <APC-A>: CD95 <PE Cy5-A>: CD28 <PE Cy7-A>: CCR7 <PE Cy5-A>: CD28 <PE Cy55-A>: CD8 <PE Cy5-A>: CD28 <PE Tx RD-A>: CD45RO <PE Cy5-A>: CD28 <PE Green laser-A>: CD127 <PE Cy5-A>: CD28

A bright Qdot 655 reagent is the problem

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SLIDE 13

PANEL ASSESSMENT USING MULTIGRAPH OVERLAYS

  • Overlay different

populations to see where subsets exist

  • Identify potential

spreading issues

00 00 00 00 00 00

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SLIDE 14

SUMMARY

  • Compensation

Ø Compensation values are arbitrary Ø All panels (but especially large) require appropriate compensation controls Ø Analyze properly transformed (and compensated) data Ø Properly compensated data reveals errors, it does not cause them

  • Spillover/Spreading

Ø Spillover/spreading error is the single most important contributor to background and loss of resolution Ø Spreading error is instruments specific Ø Spreading error is proportional to signal intensity

  • Panel Development

Ø A standardized, optimized instrument is key to successful panel development Ø Use the SSM matrix and antigen co-expression to guide marker placement Ø Titrate antibodies Ø Use appropriate controls and QC checks when assessing a new panel Ø The process is iterative and sometimes frustrating

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SLIDE 15

ACKNOWLEDGMENTS

Questions: kschwedh@fredhutch.org

Mario Roderer, NIH Stephen Perfetto, NIH Florian Mair, FH Stephen De Rosa, FH Julie McElrath, FH