V450 conjugated anti human cd8

Multicolor flow cytometry was performed using the following commercially available antibodies: phycoerythrin (PE)-conjugated anti-human PD-1, allophycocyanin (APC)-cyanine 7 (Cy7)-conjugated anti-mouse F4/80 and CD4 (BioLegend, San Diego, CA, USA), PE-conjugated anti-mouse PD-1, PerCP-Cy5.5-conjugated anti-mouse CD8a (eBioscience, San Diego, CA, USA), V500-conjugated anti-human CD3, V450-conjugated anti-human CD8, APC-H7-conjugated anti-human CD4, APC-conjugated anti-human CD69, V450-conjugated anti-mouse CD3 and CD11b, APC-conjugated anti-mouse CD69, V500-conjugated anti-mouse CD45, APC-conjugated anti PD-L1, and PE-Cy7-conjugated anti-mouse Ly6G (BD Biosciences, San Jose, CA, USA). Dead cells were excluded using the aqua fluorescent LIVE/DEAD dye (Invitrogen). Multicolor flow cytometry was performed using the LSR Fortessa, Canto II instrument (BD Biosciences). Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

Subjects included in this study were undergoing percutaneous liver biopsy for diagnostic purposes. A cell strainer (FALCON, 352350) was used to mash the biopsy samples. After that, the samples were digested by collagenase (0.05%)/Hyaluronidase (1000 U/mL) and DNase (5 U/mL) to obtain a cell suspension. Subsequently, the supernatant was removed, and the pellet was treated with an RBC lysis buffer as previously described (19 (link)).
Multicolor flow cytometry was performed using the following commercially available antibodies: phycoerythrin (PE)-conjugated anti-human CD38 and CD80, PerCP-Cy5.5-conjugated anti-human CD16, PE-cyanine 7 (Cy7)-conjugated anti-human NKG2D (BioLegend, San Diego, CA, USA), allophycocyanin (APC)-Cy7-conjugated anti-human HLA-DR, APC-conjugated anti-human CD69, V450-conjugated anti-human CD8, V500-conjugated anti-human CD4, and PerCP-Cy5.5-conjugated anti-human CD3 (BD Biosciences, San Jose, CA, USA). LIVE-DEAD Fixable Violet Dead Cell Stain Kit (Thermo Fisher Scientific, L34955) was used to discriminate dead cells from live cells. Data were recorded using an LSR Fortessa, Canto II instrument (BD Biosciences) and analyzed using the FlowJo software (TreeStar, Ashland, OR, USA). Intracellular cytokine staining was performed using a previously described protocol (20 (link)).