多色流式细胞(FACS)调补偿与圈门教学文案.ppt
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1、多色流式细胞(FACS)调补偿与圈门Compensation,Controls,and Data CollectionCompensationGoalRemove spillover signals so that subpopulation MFIs agree223-13536-00 Rev.01Compensation,Controls,and Data CollectionCompensation,Controls,and Data CollectionCompensation,Controls,and Data CollectionCompensation,Controls,and
2、Data CollectionCompensationRulesPart1The fluorescence emission spectrum of compensation controls must match the experiment reagentsthis is especially critical with tandem reagents.Compensation control negative and positive populations must be from the same cell or particle type.Example:dont use a CD
3、3 monocyte negative population with a CD3+lymphocyte positive population.623-13536-00 Rev.01Compensation,Controls,and Data CollectionCompensationRulesPart2Compensation controls must be bright enough to obtain good separation between the positive and negative populations.Compensation controls must pl
4、ace 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.723-13536-00 Rev.01Compensation,Controls,and Data CollectionBDCompBeadsUse the same antibodies as
5、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 way to create accurate single-color compensatio
6、n controls823-13536-00 Rev.01Compensation,Controls,and Data CollectionUnstainedCompensationControlTubeWhen should I use an unstained compensation control tube?In BD FACSDiva software:If using a separate unstained control,select theIncludeseparateunstainedcontroltube/wellcheckbox.If not using a separ
7、ate unstained control,clear the checkbox and for each parameter include a P3 gate for the negative population.923-13536-00 Rev.01Compensation,Controls,and Data CollectionCompensationQCBiexponential display reveals compensation problems.OverCorrectBiexponential1023-13536-00 Rev.01Compensation,Control
8、s,and Data CollectionCompensationDiscussionPointsHow often?How to QC and adjust settings?Post acquisition?Should compensation controls be treated the same as experimental samples?(example:fixed and permeabilized)1123-13536-00 Rev.01Compensation,Controls,and Data CollectionControls1223-13536-00 Rev.0
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