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. Author manuscript; available in PMC: 2019 Dec 3.
Published in final edited form as: Gut. 2016 Jan 4;66(4):724–735. doi: 10.1136/gutjnl-2015-310033

Figure 2. Detection of STAT1, TRAIL and pSTAT1-expressing NK cells of uninfected subjects and HCV-infected patients by flow cytometry.

Figure 2.

(A)Flow cytometry gating strategy. Dot plots from left to right show gating on single cells in forward scatter (FSC) area versus FSC height plots, gating on lymphocytes in FSC versus side scatter (SSC) plots, exclusion of EMA+ dead cells, CD14+ monocytes and CD19+ B cells, and gating on CD56+CD3- negative NK cells. Percentages indicate the frequency of events in the red gate relative to total number of events in the respective plot.

(B)Frequency of STAT1+ cells in the peripheral blood CD3-CD56+ NK cell population of a representative uninfected subject and a representative HCV-infected patient on day 0 and day 1 of QUAD therapy. FMO, fluorescence minus one control using all antibodies in the panel except for the anti-STAT1 A647 antibody to define the threshold of positivity. A647, alexa647.

(C)Frequency of TRAIL+ cells in the peripheral blood CD3-CD56+ NK cell population of a representative uninfected subject and a representative HCV-infected patient on day 0 and day 1 of QUAD therapy. FMO, fluorescence minus one control using all antibodies in the panel except for the anti-TRAIL PE antibody to define the threshold of positivity. PE, phycoerythrin.

(D)Frequency of pSTAT1+ cells in the peripheral blood CD3-CD56+ NK cell population of a representative uninfected subject and a representative HCV-infected patient on day 0 and day 1 of QUAD therapy. FMO, fluorescence minus one control using all antibodies in the panel except for the anti-pSTAT1 A488 antibody to define the threshold of positivity. A488, alexa488. Note that the CD56 dot plots in panels B/D (co-staining for STAT1/pSTAT1) differ from those in panel C (staining for TRAIL) because two different CD56 antibodies with different fluorochromes were used, and because of differential processing of the PBMC (fixation, permeabilisation and intranuclear staining for panels B/D; cell surface staining in panel C).