Apc cy7 anti human cd4

Thirty-two fresh tumor tissues and 11 distant normal lung tissues were collected and stored in Tissue Storage Solution (Miltenyi Biotec), and processed on the same day of sample collection. The tissues were washed with PBS until there was no visible blood on the surface, and then minced into a homogenate. Subsequently, 10 mL of type IV collagenase (Gibco) was used for digestion at 37 °C for 1 h with agitation. The resulting single-cell suspension was filtered through 70 μm cell strainer (BD Bioscience), and the supernatant was discarded after centrifugation at 500g for 10 min. After resuspension in PBS buffer, the suspension was centrifuged at 800g for 2 min, and the supernatant was discarded. This washing process was repeated twice. Fixable Viability Dye eFluor780 (eBioscience) and Zombie Yellow Viability Kit (BioLegend) were used to distinguish dead cells. Anti-human CD3 BUV395 (BD Bioscience), Anti-human CD4 APC/Cy7 (BioLegend), Anti-human CD8a Alexa Fluor700 (BioLegend), Anti-human Perforin FITC (BioLegend), and Anti-human FOXP3 PE/Cy5 (eBioscience) were used to stain cell membrane surface proteins and intracellular proteins. Samples were acquired on the BD LSR Fortessa (BD Bioscience). Flow cytometry data were analyzed using FlowJo (TreeStar). The representative gating strategies for Tregs, CD8+ T cells, and CD8+Perforin+ T cells were shown in Supplementary Figure S1.