Fluorescence Compensation. Jennifer Wilshire, PhD

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1 Fluorescence Compensation Jennifer Wilshire, PhD

2 Outline 1. Compensation Basics 2. Practical Applications -selecting comp controls (cells, stains?) 3. Multicolor Issues -spreading due to measurement error -Bi-Exponential scaling -FMO controls

3 Acknowledgements Mario Roederer - Vaccine Research Center, NIH Alan Saluk - Scripps Research Institute Carleton Stewart - Roswell Park Cancer Institute Sue DeMaggio - FloCyte Training Course Purdue Cytometry Resource

4 Importance of ACCURATE Compensation APC CD45 FL4 --> Uncompensated Compensated Over Compensated n d b n d b FL4 --> FL4 --> n b Where is the CD8 dull population?! FL3-Height --> FL3-Height --> PE-CY5-CD FL3-Height --> RPCI LFC n = negatives d = dim positives b = bright positives

5 FITC Single Stain Control FITC PE Argon Laser FL1 FL

6 Uncompensated FITC single stain control PE - no stain FITC CD3

7 Uncompensated FITC Single stain Control Unwanted signal detected in FL2 roughly 15% PE - no stain FITC CD3 Total signal detected in FL1

8 FITC Single Stain Control FITC PE Total signal detected in FL1 Unwanted signal detected in FL2 = roughly 15% FL1 FL2 True PE = Total FL2 15% FL1

9 FITC Single Stain Control Uncompensated Compensated PE - no stain FL2-15%FL1 PE - no stain FITC CD3 FITC CD3

10 Part 2: Compensation in Practice Compensation Controls - Single Stains -which cells? -which stains?

11 Compensation Controls Single Stain Controls

12 Single Stain Controls - Which cells? Does not matter as long as: The autofluorescence is the same in the negative and positive populations you are lining up. eg, Pre-gate on lymphocytes if you are using CD8 FITC as a single stain control The compensation values will be valid for ALL cell types, regardless of which type of cell is used to calculate the values. The compensation is specific for the fluorochrome, not the cell type

13 Single Stain Controls - which reagents? Use the same reagent (Ab-fluorochrome conjugate) as used in the experimental sample OR A different antibody may be substituted, as long as it is conjugated to the same fluorochrome. However

14 Single Stain Controls - which reagents? Caveats for substituting reagents: With tandem dyes (Cy5PE/Cy7PE etc.) it is necessary to use the exact same reagent spillover varies from reagent to reagent GFP, CFSE, and FITC are NOT the same fluorochrome even though they are all green! Controls should be as bright as possible As bright or brighter than the experimental stains

15 Using Beads to Compensate Antibody-capture beads Use reagent in use Lots positive Small CV, bright Some reagents won t work (IgL, EMA/PI) mix with regular comps

16 Part 3: Multicolor Issues Spreading due to measurement error -why do these populations look funny? Bi-Exponential scaling -new display options FMO controls -determine where to set your gates

17 Spreading due to Measurement Error Why do these populations look funny?

18 Multicolor Compensation Uncompensated Compensated 10 5 Lymphocytes 10 5 Lymphocytes Cy7PE-A: CD <Cy7PE-A>: CD PE-A: CD <PE-A>: CD8

19 The Actual Spread 10 5 Lymphocytes <Cy7PE-A>: CD <PE-A>: CD8

20 Bi-Exponential Scaling New Display Options

21 Bi-Exponential Scaling Compensated with New Display 10 5 Lymphocytes <Cy7PE-A>: CD <PE-A>: CD8

22 Gating controls FMO (Fluorescence Minus One)

23 Gating Controls Unstained cells or complete isotype control stains are improper controls for determining positive vs. negative expression in multi-color experiments. The best control is to stain cells with all reagents except the one of interest. FMO Control Fluorescence Minus One

24 FMO Controls FMO controls are necessary to identify cells which do or do not express a given antigen. FMO controls should be used whenever accurate discrimination is essential or when antigen expression is relatively low

25 Identifying CD4 cells with 3 colors -PBMC were stained as shown in a 3-color experiment. -Compensation was properly set for all spillovers FITC PE Cy5PE Unstained Control FMO Control Fully Stained CD3 CD8 CD3 CD4 CD PE 10 3 Unstained Bounds FMO Bounds FITC

26 3 Color Experimental Setup Example staining setup of a 3 color experiment: Tube # Description FL1 FL2 FL3 1 Experimental Sample CD3 FITC CD4 PE CD8 Cy5PE 2 Compensation Controls CD3 FITC (Single stains one for each - CD4 PE - fluorochrome used in the 4 experiment) - - CD8 Cy5PE 5 Gating Controls 6 (FMO leave out one CD3 FITC - CD8 Cy5PE fluorochrome at a time) 7 CD3 FITC CD4 PE - 8 Experimental Controls CD3 FITC CD4 PE CD8 Cy5PE 9 (fully stain healthy or CD3 FITC CD4 PE CD8 Cy5PE 10 CD3 FITC CD4 PE CD8 Cy5PE untreated samples to compare to experimental sample) * no stain added or add isotype matched control stain.

27 New Quad-Gates Log Fluor. #2 Single Positive Double Positive Single Positive Doub Positi Single Positive Log Fluorescence #1 P

28 Take Away Lessons Proper CONTROLS are essential DON T compensate by eye Use Median to adjust the populations if you must do it manually New DISPLAYS help view data intuitively

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