多色流式细胞FACS调补偿与圈门.ppt

Compensation,Controls,and Data CollectionCompensation,Controls,and Data CollectionCompensation GoalRemove spillover signals so that subpopulation MFIs agree2Compensation,Controls,and Data CollectionDoes Compensation Increase Data Spread?UncompensatedCompensatedCompensation 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 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.Spread3Compensation,Controls,and Data CollectionCytometer Settings WorkflowObtain CS&T settingsApply application settingsVerify settings with sampleCalculate compensation4Compensation,Controls,and Data CollectionCompensation 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-specificexperiment-specificlabel-specific5Compensation,Controls,and Data CollectionCompensation RulesPart 1The 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 CD3 monocyte negative population with a CD3+lymphocyte positive population.6Compensation,Controls,and Data CollectionCompensation RulesPart 2Compensation controls must be bright enough to obtain good separation 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.7Compensation,Controls,and Data CollectionBD 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 way to create accurate single-color compensation controls8Compensation,Controls,and Data CollectionUnstained 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 checkbox.If not using a separate unstained control,clear the checkbox and for each parameter include a P3 gate for the negative population.9Compensation,Controls,and Data CollectionCompensation QCBiexponential display reveals compensation problems.OverCorrectBiexponential10Compensation,Controls,and Data CollectionCompensation Discussion PointsHow often?How to QC and adjust settings?Post acquisition?Should compensation controls be treated the same as experimental samples?(example:fixed and permeabilized)11Compensation,Controls,and Data CollectionControls12Compensation,Controls,and Data CollectionChoose Appropriate ControlsWhatWhyCytometer setup controls BD CompBeadsEnsure consistent setup and compensationGating controls FMO Isotype CombinedObtain reliable gates for problem markersBiological controls Unstimulated samples Healthy donorsMake appropriate biological comparisons and conclusions13Compensation,Controls,and Data CollectionGating ControlsFluorescence-Minus-One(FMO)controlIncludes all 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 except the one of interest,which is replaced by an isotype control.Might not accurately represent the background staining of the test antibody.14Compensation,Controls,and Data CollectionFMO ExampleGated on lymphs,CD3+CD4-Gated on lymphs,CD3+CD4+Full 9-color cocktailFMOAmCyan15Compensation,Controls,and Data CollectionComparison of Gating Controls16Compensation,Controls,and Data CollectionData Collection17Compensation,Controls,and Data Collection125,000 lymphocytes collected20,000 lymphocytes collectedNumber of Events vs Measurement PrecisionCD4+T cellsCD8+T cells14 events=0.14%73 events=0.34%8 events=0.23%51 events=0.09%18Compensation,Controls,and Data CollectionStatistical Significance of ResultsDetermining the Number of Events to Collect Number of Relevant Events to Collect%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 0.10.119Compensation,Controls,and Data CollectionData Collection DiscussionStorage Gates and Stopping GatesGlobal Worksheets vs Normal WorksheetsTemplates20Compensation,Controls,and Data CollectionExperiment Setup and Record Sample Data Exercise(see workbook for additional detail)1.Create an experiment.2.Apply application settings.3.Create compensation controls,calculate and QC compensation.4.Collect sample data.Negative controlActivated sample21此课件下载可自行编辑修改，供参考！感谢您的支持，我们努力做得更好！22