1、23-13536-00 Rev. 01Compensation, Controls, and Data CollectionCompensation, Controls, and Data Collection23-13536-00 Rev. 012Compensation GoalRemove spillover signals so that subpopulation MFIs agreeCompensation, Controls, and Data Collection23-13536-00 Rev. 013Does Compensation Increase Data Spread

2、?UncompensatedCompensated Compensation corrects the MFI, but cannot remove all of the variation increase introduced by spillover. However, under normal conditions the spread in a fluorescence measurement with spillover will decrease when compensation is applied. When viewing data on log and biexpone

3、ntial plots, data spread appears to increase when compensation is appliedthis is often a visual artifact due to the non-linear scaling of the plot. SpreadCompensation, Controls, and Data Collection23-13536-00 Rev. 014Cytometer Settings WorkflowObtain CS&T settingsApply application settingsVerify set

4、tings with sampleCalculate compensationCompensation, Controls, and Data Collection23-13536-00 Rev. 015Compensation for Tandem Dyes Compensation for tandem dye conjugates can vary, even between two experiments with the same antibody. Tandem dyes require compensation that is: lot-specific experiment-s

5、pecific label-specificCompensation, Controls, and Data Collection23-13536-00 Rev. 016Compensation RulesPart 1 The fluorescence emission spectrum of compensation controls must match the experiment reagentsthis is especially critical with tandem reagents. Compensation control negative and positive pop

6、ulations must be from the same cell or particle type. Example: dont use a CD3 monocyte negative population with a CD3+ lymphocyte positive population.Compensation, Controls, and Data Collection23-13536-00 Rev. 017Compensation RulesPart 2 Compensation controls must be bright enough to obtain good sep

7、aration between the positive and negative populations. Compensation controls must place the positive population in the linear range. When using cells for compensation controls, increase the number of events to at least 10,000 per tube. Whenever MFI target values change, rerun compensation.Compensati

8、on, Controls, and Data Collection23-13536-00 Rev. 018BD CompBeads Use the same antibodies as in the experimental samples. Create bright and uniform positive fluorescence peaks. Avoid using limited sample. Beads are coated with anti-mouse kappa*.*BD CompBeads are also available coated with anti-rat k

9、appa and anti-hamster kappa.CompBeadA convenient way to create accurate single-color compensation controlsCompensation, Controls, and Data Collection23-13536-00 Rev. 019Unstained Compensation Control TubeWhen should I use an unstained compensation control tube? In BD FACSDiva software: If using a se

10、parate unstained control, select the Include separate unstained control tube/well checkbox. If not using a separate unstained control, clear the checkbox and for each parameter include a P3 gate for the negative population.Compensation, Controls, and Data Collection23-13536-00 Rev. 0110Compensation

11、QCBiexponential display reveals compensation problems.OverCorrectBiexponentialCompensation, Controls, and Data Collection23-13536-00 Rev. 0111Compensation Discussion Points How often? How to QC and adjust settings? Post acquisition? Should compensation controls be treated the same as experimental sa

12、mples? (example: fixed and permeabilized)Compensation, Controls, and Data Collection23-13536-00 Rev. 0112ControlsCompensation, Controls, and Data Collection23-13536-00 Rev. 0113Choose Appropriate ControlsWhatWhyCytometer setup controls BD CompBeadsEnsure consistent setup and compensationGating contr

13、ols FMO Isotype CombinedObtain reliable gates for problem markersBiological controls Unstimulated samples Healthy donorsMake appropriate biological comparisons and conclusionsCompensation, Controls, and Data Collection23-13536-00 Rev. 0114Gating ControlsFluorescence-Minus-One (FMO) controlIncludes a

14、ll test antibodies except the one of interest.Doesnt take background staining into account.Useful in setting gates and confirming spillover problems. Isotype controlNon-specific antibody of same isotype as the test antibody.Doesnt take spillover into account.Combined controlAll test antibodies excep

15、t the one of interest, which is replaced by an isotype control.Might not accurately represent the background staining of the test antibody.Compensation, Controls, and Data Collection23-13536-00 Rev. 0115FMO ExampleGated on lymphs, CD3+ CD4-Gated on lymphs, CD3+ CD4+Full 9-color cocktailFMOAmCyanComp

16、ensation, Controls, and Data Collection23-13536-00 Rev. 0116Comparison of Gating ControlsCompensation, Controls, and Data Collection23-13536-00 Rev. 0117Data CollectionCompensation, Controls, and Data Collection23-13536-00 Rev. 0118125,000 lymphocytes collected20,000 lymphocytes collectedNumber of E

17、vents vs Measurement PrecisionCD4+ T cellsCD8+ T cells14 events = 0.14%73 events = 0.34%8 events = 0.23%51 events = 0.09%Compensation, Controls, and Data Collection23-13536-00 Rev. 0119Statistical Significance of ResultsDetermining the Number of Events to Collect Number of Relevant Events to Collect

18、 % Background(False +) Lowest % Positive 90% power, p0.05 99% power, p0.005 0.01 0.02 260,000720,0000.01 0.05 32,00090,0000.01 12,00032,0000.02 0.05 67,000190,0000.02 16,00045,0000.03 0.05 170,000480,0000.03 0.1 23,00063,0000.04 0.1 33,00093,0000.05 0.1 52,000140,0000.06 0.1 86,000240,0000.07 0.1 160,000450,0000.08 0.2 17,00046,0000.1 0.2 26,00072,000 0.10.1Compensation, Controls, and Data Collection23-13536-00 Rev. 0120Data Collection Discussion Storage Gates and Stopping Gates Global Worksheets vs Normal Worksheets TemplatesCompensation, Contr