Fig. 1: Discordance in flow cytometric protein data and scRNAseq read counts.
From: Reply to: Questioning whether the IgM Fc receptor (FcμR) is expressed by innate immune cells
a Flow cytometry dot plots (left) illustrate the population of MerTk+ CD64+ (bold red gate) tumor mononuclear phagocytes (TMP) that were FACS-sorted for scRNAseq (top left), and this populations CD11b and F4/80 expression (bottom left). The corresponding transcript data for the markers shown in the flow plots, which was obtained from scRNAseq analysis, is overlaid on t-SNE plots for each of the four genes that encode those markers; Mertk (MerTk), Fcgr1 (CD64), Itgam (CD11b), and Adgre (F4/80) (Right). These t-SNE represent cell clusters from FACS-sorted TMP isolated from B16 melanoma tumors growing in Fcmr+/+ and Fcmr−/− mice. Gene expression is overlaid on these tSNEs to highlight differential read counts for each of the four genes, where a UMI of 0 is illustrated in blue. b Histograms illustrating the distributions of raw UMI counts per cell for those same four genes (Mertk, Fcgr1, Itgam, and Adgre) which were stained at the protein level during FACS-sorting of TMP. Mean count values are denoted by a vertical red dashed line. c and d (Left) The bicolor contour plots illustrate surface receptors Ly-6c and CD11c backgated from histogram plots (middle), where the Ly-6chi is gated in orange (top) and the CD11chi is gated in blue (bottom). Apparent from the backgating (left), both surface receptors Ly-6c (Ly6c1) and CD11c (Itgax) are expressed at the protein level on the TMP that were sorted for scRNAseq. (Right) tSNE expression-overlaid plots for Ly6c1 (top right) and Itgax (bottom right) show zero and near zero cells with transcript for these receptors. UMI of 0 is illustrated in blue. d Histograms illustrating the distribution of UMI for Ly6c1 and Itgax, as in b above.
