流式细胞(FACS)多色分析.ppt

23-13536-00 Rev.01,Multicolor Analysis,23-13536-00 Rev.01,2,Multicolor Analysis Strategies,Plot typesHierarchical gatingPopulation hierarchiesBack gating,23-13536-00 Rev.01,3,Use Different Plot Types,Histogramsusing histograms can help discriminate between two populations.,Dot Plot:hard to discriminate populations,Dot Plot:displaying fewer events improves discrimination,Histogram:population boundary is evident,23-13536-00 Rev.01,4,Use Different Plot Types,Contour Plotsusing contour plots can:Help locate population means.Indicate a need for biexponential scaling.,Dot Plot:hard to see populations bunched on axis,Contour Plot:shows that biexponential display might be needed,Biexponential Contour Plot:gives best display of populations,23-13536-00 Rev.01,5,Hierarchical Gating,23-13536-00 Rev.01,6,Hierarchical Gating,What went wrong here?,Correct Hierarchy,23-13536-00 Rev.01,7,Back Gating,Researcher wants to discriminate Tregs based on CD25 and CD127 expression.Need to ensure a gate includes the entire FOXP3+population.,23-13536-00 Rev.01,8,Additional Analysis Strategies,Biexponential scalingEvents to displayHinged and offset quadrant gatesDerived(boolean)gatesSnap-to gatesStatisticsAnalysis templates,23-13536-00 Rev.01,9,ICS Data Analysis Results,Information that can be retrieved:Number of collected events,number of live CD3+cells%of main populations:%CD3+,CD4+,CD8+%of nave(CD45RA+)and memory(CD45RA)CD4+and CD8+cells%CD25+CD4+cells(Tregs)For the cytokines:%of CD4+and CD8+making 1 or 2 cytokines,Analysis Exercise,23-13536-00 Rev.01,10,ICS Panel Analysis,