Histogram overlays of antigen expression by CD8 + T N across the 5 experiments following biexponential transformation. Biexponential transformation normalizes background fluorescence across independent experiments. Dotted horizontal bar indicates threshold of positivity of CD45RO (y axis). CD45RO in CD8 + T N‐gated cells from one representative donor. b) Fluorescence levels of CD56, HLA‐DR, CD4 and CD45RO in CD8+ T N cells from the two samples included in the analysis. a) tSNE representation of total events and of T N‐gated CD8 + T cells (defined as CD4 –CD45RO –CCR7 +). tSNE analysis included the following parameters: CD56 PE‐Cy5, HLA‐DR APC‐H7, CD4 APC and CD45RO PerCP‐Cy5.5. PBMCs from two donors, stained in the same day with the same mix of multiple fluorescently‐conjugated monoclonal antibodies, were acquired on a FACSAria cell sorter, then concatenated for further analysis by tSNE in R. Different tSNE displays are associated with different background fluorescence values. The black arrow indicates subpopulations identified following randomization of negative events below a threshold of 100. Dotted horizontal bar indicates threshold of positivity of CD25, CD57 and CD69 expression (y axis). b) Overlay of tSNE1 ans tSNE2 axes obtained as in a. Each run shows pooled CD8 + T cells from 3 different donors for simplicity (3,000 cells each).
Numbers in plots indicate the percentage of cells identified by the arbitrary gate. a) tSNE map of 27‐parameter flow cytometry data from 5 replicate experiments following randomization of background at 100. Background randomization of negative events improves visualization of high‐dimensional FACS data. Channel labels refer to the laser source (UV: ultraviolet V: violet B: blue YG: yellow/green R: red) and the median of wavelength detection by the bandpass filter. a) Background and b) positive signals across independent experiments as revealed by unstained and single‐peak rainbow beads, respectively. Machine performance across independent experiments. Matrix overlay of antigen expression between run 1 and run 5. Similar distribution of compensated data between independent experiments. The grey bar in the background represents the interquartile range of the distribution. Each dot represents a single independent experiment. b) Frequency of CD8 + T cells expressing a given antigen in 3 different healthy donors (HD). a) Gating strategy of the identification of CD8 + T cells in peripheral blood used in this study. 27‐parameter FACS is reproducible across independent experiments.
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