多色流式细胞(FACS)调补偿与圈门

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1、Compensation,Controls,and Data CollectionCompensation,Controls,and Data Collection2Compensation GoalRemove spillover signals so that subpopulation MFIs agreeCompensation,Controls,and Data Collection3Does Compensation Increase Data Spread?UncompensatedCompensated Compensation corrects the MFI,but can

2、not 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 ap

3、pliedthis is often a visual artifact due to the non-linear scaling of the plot.SpreadCompensation,Controls,and Data Collection4Cytometer Settings WorkflowObtain CS&T settingsApply application settingsVerify settings with sampleCalculate compensationCompensation,Controls,and Data Collection5Compensat

4、ion 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-specific label-specificCompensation,Controls,and Data Collection6Compensation RulesPart 1 The fluorescence emission

5、 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

6、population.Compensation,Controls,and Data Collection7Compensation RulesPart 2 Compensation 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 compensa

7、tion controls,increase the number of events to at least 10,000 per tube.Whenever MFI target values change,rerun compensation.Compensation,Controls,and Data Collection8BD CompBeads Use the same antibodies as in the experimental samples.Create bright and uniform positive fluorescence peaks.Avoid using

8、 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 controlsCompensation,Controls,and Data Collection9Unstained Compensation Control TubeWhen s

9、hould 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 pop

10、ulation.Compensation,Controls,and Data Collection10Compensation QCBiexponential display reveals compensation problems.OverCorrectBiexponentialCompensation,Controls,and Data Collection11Compensation Discussion Points How often?How to QC and adjust settings?Post acquisition?Should compensation control

11、s be treated the same as experimental samples?(example:fixed and permeabilized)Compensation,Controls,and Data Collection12ControlsCompensation,Controls,and Data Collection13Choose Appropriate ControlsWhatWhyCytometer setup controls BD CompBeadsEnsure consistent setup and compensationGating controls

12、FMO Isotype CombinedObtain reliable gates for problem markersBiological controls Unstimulated samples Healthy donorsMake appropriate biological comparisons and conclusionsCompensation,Controls,and Data Collection14Gating ControlsFluorescence-Minus-One(FMO)controlIncludes all test antibodies except t

13、he 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

14、is replaced by an isotype control.Might not accurately represent the background staining of the test antibody.Compensation,Controls,and Data Collection15FMO ExampleGated on lymphs,CD3+CD4-Gated on lymphs,CD3+CD4+Full 9-color cocktailFMOAmCyanCompensation,Controls,and Data Collection16Comparison of G

15、ating ControlsCompensation,Controls,and Data Collection17Data CollectionCompensation,Controls,and Data Collection18125,000 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%Compen

16、sation,Controls,and Data Collection19Statistical 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

17、,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 Collection20Data Collection Discussion Storage Gates and Stopping G

18、ates Global Worksheets vs Normal Worksheets TemplatesCompensation,Controls,and Data Collection21Experiment Setup and Record Sample Data Exercise(see workbook for additional detail)Create an experiment.Apply application settings.Create compensation controls,calculate and QC compensation.Collect sample data.Negative control1.Activated sample个人观点供参考，欢迎讨论