The HCC cells were co-cultured with activated T cells for 48h at the ratio of 1:1. Brefeldin A (BD Biosciences) was pretreated with the mixed cells for 6 h. After permeabilizing using fixation and permeabilization reagents (Biolegend 426803), the cells were stained with anti-human CD8-BV510 antibodies (Biolegend 344731), anti-human INF-r-BV421 (Biolegend 506537) and anti-human TNF-a-PE (Biolegend 502908) for 20 mins. Then the samples were ready for flow cytometry.
To analyze membrane PD-L1, HCC cells were dissociated with trypsin-EDTA solution and stained with anti-human CD274 PE antibodies (Biolegend 329705) and incubated for 20 min. Then the cells were harvested for flow analysis.
For PD-L1 flow cytometry from patient samples, we prepared a single-cell suspension from patient samples using MagicFilter and MagicVajra (Bozhentech B160103). Then the suspended cells were stained with APC anti-human CD45 antibodies (Biolegend 304012), Brilliant Violet 510 anti-human CD8 antibodies (Biolegend 344732), FITC anti-human CD3 antibodies (Biolegend 300406), PE anti-human CD274 antibodies (Biolegend 329706), and 7-AAD Viability Staining (Biolegend 420404). After a 20 mins staining, cells were re-suspended in 300 μL of PBS for flow analysis.
To analyze membrane PD-L1, HCC cells were dissociated with trypsin-EDTA solution and stained with anti-human CD274 PE antibodies (Biolegend 329705) and incubated for 20 min. Then the cells were harvested for flow analysis.
For PD-L1 flow cytometry from patient samples, we prepared a single-cell suspension from patient samples using MagicFilter and MagicVajra (Bozhentech B160103). Then the suspended cells were stained with APC anti-human CD45 antibodies (Biolegend 304012), Brilliant Violet 510 anti-human CD8 antibodies (Biolegend 344732), FITC anti-human CD3 antibodies (Biolegend 300406), PE anti-human CD274 antibodies (Biolegend 329706), and 7-AAD Viability Staining (Biolegend 420404). After a 20 mins staining, cells were re-suspended in 300 μL of PBS for flow analysis.