Transcription factor fixation permeabilization buffer

CD8 + T cells from tumor-infiltrating lymphocytes were isolated using flow cytometry-assisted cell sorting. The purity of the sorted cells was >95 %. To detect cytokine production, lymphocytes were stimulated for 2 h in the presence of a cell-stimulating mixture (eBioscience, USA, 00-4975-93) and then stained with the surface markers anti-LAG-3 (BioLegend, USA, Cat. No. 125214) and anti-PD-1 (BioLegend, Cat. No. 135206), and CTLA4 (BioLegend, Cat. No. 106308) at a 1:100 dilution in PBS containing 1 % FBS. Following a 30 min incubation on ice, a cell fixation/permeabilization kit (BD Biosciences, USA, Cat. No. 554714) and a transcription factor fixation/permeabilization buffer (BioLegend, Cat. No. 424401) were employed for intracellular cytokine and nuclear transcription factor labeling, respectively. Anti-IFN-γ (BioLegend, USA, Cat. No. 505810), anti-TNF-α (BioLegend, Cat. No. 506306), and anti-TOX (Miltenyi Biotec, Germany, Cat. No. 130-120-337) were diluted 1:50 in permeabilization buffer and incubated overnight at 4 °C in the presence of Anti-IFN-γ and anti-TNF-α(BioLegend, USA). FlowJo software (Tree Star, USA) was used to analyze FACSCelesta (BD, USA) flow cytometry data. All analyses were performed using GraphPad Prism 8 (GraphPad Software, USA) and t-tests. P < 0.05 was deemed statistically significant.

Gastric cancer tissues were cut into small pieces, digested with collagenase/DNase I, and filtered through 70 μm cell strainers to produce a single cell suspension.
First, live/dead fixable was used as a live/dead marker to gate out the dead cells. The samples were then surface-labeled with fluorochrome-conjugated antibodies against CD3 and CD4 for 30 min, and the transcription factor fixation/permeabilization buffer (Biolegend) was used for FOXP3 staining.
Samples were analyzed by flow cytometry (BD), and data were analyzed using FlowJo V10 software.