##Example1 library(flowCore) library(flowDensity) data_dir <- system.file("extdata", package = "flowDensity") load(list.files(pattern = 'sampleFCS_1', data_dir, full = TRUE)) f sngl <- flowDensity(f,channels = c("FSC-A","FSC-H"),position = c(F,F), percentile =c(.99999,.99999),use.percentile = c(T,T), ellip.gate = T,scale = .99 ) plotDens(f,c(1,2)) lines(sngl@filter,type="l") ###Example2 bcell <- flowDensity(sngl, channels=c(9, 3), position=c(FALSE, NA)) plot(getflowFrame(sngl), bcell) ###Example3 CD19pCD20n <- flowDensity(obj=bcell, channels=c(8, 6), position=c(T,F)) plasmablasts <- flowDensity(obj=CD19pCD20n, channels=c(5, 12), position=c(T, T)) plotDens(getflowFrame(CD19pCD20n), plasmablasts@channels, pch=19) points(plasmablasts@filter, type='l', col=2, lwd=2) ##Removing margin f <- nmRemove(f, c("FSC-A", "SSC-A")) ##Multiple step gating load(list.files(pattern = 'sampleFCS_2', data_dir, full = TRUE)) f2 channels <- c("V500-A", "SSC-A") # First call to flowDensity tmp.cp1 <- flowDensity(obj=f2, channels=channels, position=c(TRUE, FALSE), percentile=c(0.25, NA)) # Second call to flowDensity tmp.cp2 <- flowDensity(obj=tmp.cp1, channels=channels, position=c(TRUE, FALSE), gates=c(FALSE, NA), percentile=c(NA, 0.85)) # Final call to flowDensity lymph <- flowDensity(obj=f2, channels=channels, position=c(TRUE, FALSE), gates=c(tmp.cp1@gates[1], tmp.cp2@gates[2]), ellip.gate=TRUE, scale=.99) plot(f2, tmp.cp1) plot(f2, tmp.cp2) plotDens(f2, channels=channels) points(lymph@filter, type="l", col=2, lwd=2) ##Using FMOs load(list.files(pattern = 'sampleFCS_3.Rdata', data_dir, full = TRUE)) f3 load(list.files(pattern = 'sampleFCS_3_FMO', data_dir, full = TRUE)) f3.fmo f3.gated <- flowDensity(obj=f3, channels=c('BV421-A', 'FSC-A'), position = c(TRUE, NA),use.control = c(TRUE, F) , control = c(f3.fmo, NA)) f3.fmo.gated <- flowDensity(obj=f3.fmo, channels=c('BV421-A', 'FSC-A'), position=c(TRUE, NA), gates=c(f3.gated@gates[1], NA)) plot(f3.fmo, f3.fmo.gated) plot(f3, f3.gated) ##Using percentile f3.gated.98p <- flowDensity(obj=f3, channels=c('BV421-A', 'FSC-A'), position = c(TRUE, NA),use.percentile = c(TRUE, NA), percentile = 0.98, use.control = c(TRUE, FALSE), control = c(f3.fmo, NA)) f3.fmo.gated.98p <- flowDensity(obj=f3.fmo, channels=c('BV421-A', 'FSC-A'), position = c(TRUE, NA), gates=c(f3.gated.98p@gates[1], NA)) plot(f3.fmo, f3.fmo.gated.98p) plot(f3, f3.gated.98p) #Using other arguments load(list.files(pattern = 'sampleFCS_2', data_dir, full = TRUE)) thresholds <- deGate(obj = f2,channel = 9) #Percentile default is .95, which can be changed thresholds.prcnt <- deGate(f2,channel = 9,use.percentile=T,percentile=.3) thresholds.lo <- deGate(f2,channel = 9,use.upper=T,upper=F,alpha = .9) thresholds.hi <- deGate(f2,channel = 9,use.upper=T,upper=T,alpha = .9) plotDens(f2,c(9,12)) abline(v=c(thresholds,thresholds.prcnt,thresholds.lo,thresholds.hi),col=c(1,2,3,4))