Figure 3. Reducing staining volume primarily affects highly expressed markers.
Comparison of peripheral blood mononuclear cell samples stained in 50 µL (same sample as dilution factor [DF] 4 in Figure 1) or 25 µL volume at DF4. (A) Summarized unique molecular identifier (UMI) counts within cell-containing droplets segmented by the individual antibodies colored by their concentration. (B, C) Change in antibody-derived tag signal for each antibody by reducing staining volume from 50 to 25 µL. Individual antibodies are colored by their concentration. Quantified by (B) sum of UMIs within cell-containing droplets assigned to each antibody and (C) 90th percentile UMI count within the cell type with most abundant expression (the assayed cell type is annotated to the right). (D) Titration plot (marker UMI count vs. normalized cell rank) for CD31 signal response when reducing staining volume from 50 µL to 25 µL. Histogram depicts distribution of UMIs at each condition. Barcodes to the right depict cell-type occurrence at the corresponding rank to visualize cell specificity of the antibody. Numbers on top of the small bar plot denote total UMI count assigned to CD31 within cell-containing droplets from each condition. (E, F) Non-normalized UMI counts visualized on t-distributed stochastic neighbor embedding (tSNE) plots of an affected (CD31; E) or an unaffected (CD8; F) marker by the reduction in cell density. Dashed line indicates the region where expression levels vary between volumes. Titration plots for all markers can be found in Figure 3—figure supplements 1 and 2.
Figure 3—figure supplement 1. Response of individual antibodies to reduction in staining volume.
Figure 3—figure supplement 2. Response of individual antibodies to reduction in staining volume.
