Anti human granzymeb

Peripheral blood mononuclear cells (PBMC) were extracted from 4ml of venous blood obtained from HCC patients for flow cytometry analysis. We used the anti-human CD8 (Biolegend) marker to identify CD8⁺T cells. Notch1⁺CD8⁺T cells were identified using the anti-human Notch1 (BD) antibody. Additionally, we assessed the expression of co-inhibitory molecules on the cell surface using anti-human TIGIT (BD), anti-human TIM-3 (BD), anti-human CD39 (Biolegend), and anti-human PD-1 (BD) antibodies. Furthermore, we evaluated the killing function of the cells by detecting the expression of anti-human Perforin (Biolegend) and anti-human GranzymeB (BD) antibodies.

Fresh cells were isolated from spleen, thymus, bone marrow, or whole blood from mice at ages indicated. T and B cells were enriched using EasySep kit (Stem Cell Technologies, Vancouver, BC, Canada). Flow optimizations and protocols were used as previously described [61 (link)]. Gating strategy and graphs from representative C57BL/6 and Eµ-TCL1 whole blood are shown in the supplementary materials. All gates were set using fluorescent antibody controls prepared for each individual experiment. Flow cytometry antibodies used in this study include: anti-mouse CD45 (BD #559,864), anti-mouse CD5 (BD #553,023), anti-mouse CD19 (BD #562,701), anti-mouse CD45R/B220 (BD #553,088), anti-human CD3 (BD #555,335), anti-human CD19 (BD #562,440), anti-human CD2 (BD #555,326), anti-human CD28 (BD #555,729), anti-human Granzyme B (BD #561,142), anti-human IFN-gamma (BD #554,551). All data were analyzed using Kaluza v2.0 software (Becton Dickinson).