Fig. 2.

NOckout^Fn detects NO in mouse primary microglia and in alveolar macrophages. (A) Representative confocal images of NOckout from LPS (1 μg/mL) primed J774A.1 macrophages in A647 (R) and DAR (G) channels in the absence (Upper) or presence (Lower) of NOS2 inhibitor 1400W. G/R intensities are represented as heat maps. (B) Representative confocal images of NOckout^mCpG from mouse primary microglia. Cells were incubated with NOckout^mCpG (500 nM) in the absence (Upper) or presence (Lower) of 1400W for 120 min in DMEM, imaged in DAR(G) and A647(R) channels, and converted into G/R heat maps. (C) Representative heat map (G/R) images of NOckout-, NOckout^iRNA-, and NOckout^RNA-treated J774A.1 cells. (D) Violin plot of the distribution of G/R values of ∼200 individual endosomes (n = 30 cells) of J774A.1 cells treated with VAS2870, ABAH, and 1400W in the presence and absence of LPS. (E) Violin plot of the distribution of G/R values of ∼100 individual endosomes (n = 20 cells) in primary microglia treated with NOckout^mCpG and NOckout^mGpC. (F) Violin plot of the distribution of G/R values of ∼200 individual endosomes (n = 30 cells) in J774A.1 macrophages treated with NOckout^RNA variants in the presence and absence of 1400W. All experiments were performed in triplicate. (Scale bar, 10 μm.) P values are obtained using Kruskal−Wallis statistical test across the dataset.