多色流式细胞(FACS)调补偿与圈门课件

1、Compensation, Controls, and Data Collection,1,PPT学习交流,Compensation Goal,Remove spillover signals so that subpopulation MFIs agree,2,PPT学习交流,Does Compensation Increase Data Spread?,Uncompensated,Compensated,Compensation corrects the MFI, but cannot remove all of the variation increase introduced by s

2、pillover. 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-

3、linear scaling of the plot.,Spread,3,PPT学习交流,Cytometer Settings Workflow,Obtain CS&T settings,Apply application settings,Verify settings with sample,Calculate compensation,4,PPT学习交流,Compensation for Tandem Dyes,Compensation for tandem dye conjugates can vary, even between two experiments with the sa

4、me antibody. Tandem dyes require compensation that is: lot-specific experiment-specific label-specific,5,PPT学习交流,Compensation RulesPart 1,The fluorescence emission spectrum of compensation controls must match the experiment reagentsthis is especially critical with tandem reagents. Compensation contr

5、ol 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.,6,PPT学习交流,Compensation RulesPart 2,Compensation controls must be bright enough to obtain good separation between the posi

6、tive 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.,7,PPT学习交流,BD CompBeads,Use the sa

7、me 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.,CompBead,A convenient way to create accurate s

8、ingle-color compensation controls,8,PPT学习交流,Unstained Compensation Control Tube,When 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 unstaine

9、d control, clear the checkbox and for each parameter include a P3 gate for the negative population.,9,PPT学习交流,Compensation QC,Biexponential display reveals compensation problems.,Over,Correct,Biexponential,10,PPT学习交流,Compensation Discussion Points,How often? How to QC and adjust settings? Post acqui

10、sition? Should compensation controls be treated the same as experimental samples? (example: fixed and permeabilized),11,PPT学习交流,Controls,12,PPT学习交流,Choose Appropriate Controls,13,PPT学习交流,Gating Controls,Fluorescence-Minus-One (FMO) control Includes all test antibodies except the one of interest. Doe

11、snt take background staining into account. Useful in setting gates and confirming spillover problems. Isotype control Non-specific antibody of same isotype as the test antibody. Doesnt take spillover into account. Combined control All test antibodies except the one of interest, which is replaced by

12、an isotype control. Might not accurately represent the background staining of the test antibody.,14,PPT学习交流,FMO Example,FMO AmCyan,15,PPT学习交流,Comparison of Gating Controls,16,PPT学习交流,Data Collection,17,PPT学习交流,125,000 lymphocytes collected,20,000 lymphocytes collected,Number of Events vs Measurement

13、 Precision,CD4+ T cells,CD8+ T cells,14 events = 0.14%,73 events = 0.34%,8 events = 0.23%,51 events = 0.09%,18,PPT学习交流,Statistical Significance of Results,Determining the Number of Events to Collect,Number of Relevant Events to Collect,% Background (False +),Lowest % Positive,90% power, p0.05,99% po

14、wer, p0.005,0.01,0.02,260,000,720,000,0.01,0.05,32,000,90,000,0.01,12,000,32,000,0.02,0.05,67,000,190,000,0.02,16,000,45,000,0.03,0.05,170,000,480,000,0.03,0.1,23,000,63,000,0.04,0.1,33,000,93,000,0.05,0.1,52,000,140,000,0.06,0.1,86,000,240,000,0.07,0.1,160,000,450,000,0.08,0.2,17,000,46,000,0.1,0.2,26,000,72,000,0.1,0.1,19,PPT学习交流,Data Collection Discussion,Storage Gates and Stopping Gates Global Worksheets vs Normal Worksheets Templates,20,PPT学习交流,Experiment Setup and Record Sampl