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. 2020 Aug 25;11:1903. doi: 10.3389/fimmu.2020.01903

Figure 3.

Figure 3

VEGFA expression is not enhanced by ILC1-like phenotype conversion. (A) Unsuccessful detection of intracellular VEGF-A production by flow cytometry using the anti-VEGF-A clone 23410. Cells were cultured in with or without hypoxia (1% O2) for 3 days. (B) Representative fluorescence in-situ hybridization (FISH) flow cytometry plot quantifying VEGFA gene expression by NK cells cultured in normoxia (black) or hypoxia at 4% O2 (blue) or 1% O2 (red). (C) Fluorescent microscopy detection of FISH VEGFA mRNA probes (red) in normoxic or hypoxic (1% O2) NK cells counterstained with DAPI (blue). (D) Representative FISH flow cytometry for B2M control gene expression under the culture conditions detailed in (B). (E) Representative flow cytometry plot of CD9 and CD103 expression after 3 days in culture with TGF-β and hypoxia, demonstrating gating for CD9−CD103− (black), CD9+CD103− (blue), CD9−CD103+ (red), and CD9+CD103+ (purple) NK cell subsets. (F) Representative flow cytometry histograms demonstrating FISH-detected VEGFA expression under normoxia and hypoxia for the subsets gated in (E). (G) Quantification of FISH-VEGFA expression for the subsets gated in (E) on NK cells cultured for 3 days with TGF-β and hypoxia (1% O2). (MFI-FMO = median fluorescence intensity less fluorescence minus one control). n = 3. ns = not significant by ANOVA.