High-throughput imaging of mRNA at the single-cell level in human primary immune cells

hcHCR allows cell gating based on gene expression. Human primary T cells (total CD3+ T cells) were obtained from a healthy donor and hcHCR was performed with CD4 and FOXP3 as the target mRNAs. The hcHCR image output was converted to FCS format, and histograms of the spots-per-cell counts were displayed in the flow cytometry software FlowJo. Gates were created for CD4-positive (defined as cells with ≥1 CD4 spots/cell) or CD4-negative cells (top), and FOXP3 expression was displayed for the events in each gate (bottom).