Simultaneous quantification of transcript abundance for multiple genes. Single-cell level measurement of gene expression changes for three genes, TNF (cyan), IL1B (magenta), and PGK1 (yellow) assayed in the same reaction in primary human monocytes stained with DAPI (gray). (A) Time-series showing HCR signals for TNF, IL1B, and PGK1 following vehicle or LPS (1 ng/mL) stimulation at 15-, 30-, and 60-min time points. Magnification: 60×. Scale bar for full FOV: 20 microns. Scale bar for inset: 10 microns. (B) Scatter plots of TNF and IL1B gene expression, quantified as the number of HCR spots per cell. Cells were stimulated with vehicle or LPS for 30 min before hcHCR. Least-squares regression lines are in red. r = Pearson correlation coefficient. (C) Appearance of large and bright nuclear IL1B or TNF HCR spots in the cell nucleus upon LPS stimulation (arrowheads). Magnification: 60×. Scale bar: 10 microns. (D) Line plots of gene expression changes, measured simultaneously by hcHCR, for TNF, IL1B, and PGK1 before and after LPS (1 ng/mL) stimulation at different time points. Results of three independent experiments performed on cells from unrelated healthy human donors. P-values are from a Kruskal–Wallis one-way ANOVA on ranks. (E) Scatter plots of IL1B and TNF expression after LPS stimulation at six concentrations (0, 0.01, 0.1, 1, 10, or 100 ng/mL) and five time points (0, 15, 30, 60, or 120 min). (F) Line plots of gene expression, measured simultaneously by hcHCR, for TNF, IL1B, and PGK1 after 15 min of LPS (1 ng/mL) stimulation at different concentrations. Results of three independent experiments performed on cells from unrelated healthy human donors. P-values are from a Kruskal–Wallis test (one-way ANOVA on ranks).
