Cd38 pe texas red

For phenotypic analysis of NK cells, PBMC were stained with the following fluorochrome conjugated antibodies or isotype matched controls: CD3-Cy5.5/PerCP or CD3/Pe-Cy7, (eBioscience, Hatfield, UK), CD56-PE Texas Red (Beckman Coulter, High Wycombe, UK), CD56-FITC, CD16-APC Cy7, HLA-DR V500, CXCR3-Cy.5./PerCP, CD57-BV605, NKp46-V450, TRAIL-PE, (BD Biosciences, Oxford, UK), TRAIL-BV421, CD62L-AF700, CXCR6-PE, CD69-APC, CCR7-BV421, KLRG1-FITC, (Biolegend, London, UK), NKG2A-APC, NKG2C-Cy5.5/PerCP, NKG2D-PE (R&D systems, Abingdon, UK), NKp30-APC, NKp44-PE (Miltenyi Biotec, Surrey, UK), in the presence of fixable live/dead stain (Invitrogen, Paisley, Scotland). For phenotypic analysis of T cells PBMC were stained with the fluorochrome conjugated antibodies or isotype matched controls: CD3/Pe-Cy7, CD8-AF700, CD4-APC Cy7 (eBioscience, Hatfield, UK), CD38-PE Texas Red, CD14-V500, CD19-V500 (Biolegend, London, UK). Flourescence minus-one (FMOs) were used for gating purposes for all flourochromes; an example of the gating strategy is shown in S1A Fig. Cells were acquired on a FACS LSRII multicolour flow cytometer (Beckton Dickinson) and analysed using Flow Jo analysis software (Tree star, Ashland, OR, USA). In addition to percentage, absolute numbers of NK cell subsets were calculated by multiplying their percent by total lymphocyte count.

Patients were tested for their HLA-A2 status and PBMC from HLA-A2+ patients were stimulated with peptides representing HLA-A2-restricted HBV epitopes (HBVenv: FLLTRILTI, WLSLLVPFV, LLVPFVQWFV, GLSPTVWLSV; HBVcore: FLPSDFFPSV; HBVpol: GLSRYVARL, KLHLYSHPI) or the CMV pp65-encoded NLVPMVATV epitope (Proimmune). Virus-specific cells were identified by multicolour flow-cytometry (BD LSR II): surface staining with CD3/Pe-Cy7, CD8-AF700, CD4-APC Cy7 (eBioscience, Hatfield, UK), CD38-PE Texas Red, CD14-V500, CD19-V500 (Biolegend, London, UK) in the presence of fixable live/dead stain (Invitrogen).