多色流式细胞(FACS)调补偿与圈门.ppt

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1、23-13536-00 Rev. 01,Compensation, Controls, and Data Collection,23-13536-00 Rev. 01,2,Compensation Goal,Remove spillover signals so that subpopulation MFIs agree,23-13536-00 Rev. 01,3,Does Compensation Increase Data Spread?,Uncompensated,Compensated,Compensation corrects the MFI, but cannot remove a

2、ll 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 appliedthi

3、s is often a visual artifact due to the non-linear scaling of the plot.,Spread,23-13536-00 Rev. 01,4,Cytometer Settings Workflow,Obtain CS&T settings,Apply application settings,Verify settings with sample,Calculate compensation,23-13536-00 Rev. 01,5,Compensation for Tandem Dyes,Compensation for tand

4、em dye conjugates can vary, even between two experiments with the same antibody. Tandem dyes require compensation that is: lot-specific experiment-specific label-specific,23-13536-00 Rev. 01,6,Compensation RulesPart 1,The fluorescence emission spectrum of compensation controls must match the experim

5、ent 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.,23-13536-00 Rev. 01,7,Compensation RulesP

6、art 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 compensation controls, increase the number of events to at least 10,000 per tu

7、be. Whenever MFI target values change, rerun compensation.,23-13536-00 Rev. 01,8,BD CompBeads,Use 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 ava

8、ilable coated with anti-rat kappa and anti-hamster kappa.,CompBead,A convenient way to create accurate single-color compensation controls,23-13536-00 Rev. 01,9,Unstained Compensation Control Tube,When should I use an unstained compensation control tube? In BD FACSDiva software: If using a separate u

9、nstained 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.,23-13536-00 Rev. 01,10,Compensation QC,Biexponential display reveals compensation pr

10、oblems.,Over,Correct,Biexponential,23-13536-00 Rev. 01,11,Compensation Discussion Points,How often? How to QC and adjust settings? Post acquisition? Should compensation controls be treated the same as experimental samples? (example: fixed and permeabilized),23-13536-00 Rev. 01,12,Controls,23-13536-0

11、0 Rev. 01,13,Choose Appropriate Controls,23-13536-00 Rev. 01,14,Gating Controls,Fluorescence-Minus-One (FMO) control Includes all test antibodies except the one of interest. Doesnt take background staining into account. Useful in setting gates and confirming spillover problems. Isotype control Non-s

12、pecific 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 an isotype control. Might not accurately represent the background staining of the test antibody.,23-13536-00 Rev. 01,15,FMO

13、 Example,FMO AmCyan,23-13536-00 Rev. 01,16,Comparison of Gating Controls,23-13536-00 Rev. 01,17,Data Collection,23-13536-00 Rev. 01,18,125,000 lymphocytes collected,20,000 lymphocytes collected,Number of Events vs Measurement Precision,CD4+ T cells,CD8+ T cells,14 events = 0.14%,73 events = 0.34%,8

14、events = 0.23%,51 events = 0.09%,23-13536-00 Rev. 01,19,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% power, p0.005,0.01,0.02,260,000,720,000,0.01,0.05,32,000,90,000,0

15、.01,12,000,32,000,0.02,0.05,67,000,190,000,0.02,16,000,45,000,0.03,0.05,170,000,480,000,0.03,0.1,23,000,63,000,0.04,0.1,33,000,93,000,0.05,0.1,52,000,140,000,0.06,0.1,86,000,240,000,0.07,0.1,160,000,450,000,0.08,0.2,17,000,46,000,0.1,0.2,26,000,72,000,0.1,0.1,23-13536-00 Rev. 01,20,Data Collection D

16、iscussion,Storage Gates and Stopping Gates Global Worksheets vs Normal Worksheets Templates,23-13536-00 Rev. 01,21,Experiment 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 control Activated sample,