多色流式细胞FACS调补偿与圈门.pptx
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1、23-13536-00 Rev.011Compensation GoalRemove spillover signals so that subpopulation MFIs agree第1页/共21页23-13536-00 Rev.012Does Compensation Increase Data Spread?UncompensatedCompensatedCompensation corrects the MFI,but cannot remove all of the variation increase introduced by spillover.However,under n
2、ormal conditions the spread in a fluorescence measurement with spillover will decrease when compensation is applied.When viewing data on log and biexponential plots,data spread appears to increase when compensation is appliedthis is often a visual artifact due to the non-linear scaling of the plot.S
3、pread第2页/共21页23-13536-00 Rev.013Cytometer Settings WorkflowObtain CS&T settingsApply application settingsVerify settings with sampleCalculate compensation第3页/共21页23-13536-00 Rev.014Compensation for Tandem Dyes Compensation for tandem dye conjugates can vary,even between two experiments with the same
4、 antibody.Tandem dyes require compensation that is:lot-specificexperiment-specificlabel-specific第4页/共21页23-13536-00 Rev.015Compensation RulesPart 1The fluorescence emission spectrum of compensation controls must match the experiment reagentsthis is especially critical with tandem reagents.Compensati
5、on control negative and positive populations must be from the same cell or particle type.Example:dont use a CD3 monocyte negative population with a CD3+lymphocyte positive population.第5页/共21页23-13536-00 Rev.016Compensation RulesPart 2Compensation controls must be bright enough to obtain good separat
6、ion 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.第6页/共21页23-13536-00
7、 Rev.017BD CompBeadsUse 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 kappa and anti-hamster kappa.CompBeadA convenient
8、way to create accurate single-color compensation controls第7页/共21页23-13536-00 Rev.018Unstained Compensation Control TubeWhen should I use an unstained compensation control tube?In BD FACSDiva software:If using a separate unstained control,select the Include separate unstained control tube/well checkb
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