Zombie nir fixable viability dye kit

Cryopreserved single-cell suspensions from GBM tumor tissue (and peripheral blood mononuclear cells (PBMCs) for use as controls) were thawed. In the bipositive SOX2⁺CD68⁺ TAMs identification experiment, samples were stained for viability with Zombie NIR Fixable Viability Dye Kit (1:500 dilution, BioLegend, #423101) at 25°C for 20 min in the dark. Samples were then washed in phosphate-buffered saline (PBS) supplemented with 2% fetal bovine serum (FBS), before staining with a master mix of surface antibodies (Alexa Fluor 647 anti-human CD68 Antibody, BioLegend, #333820), and in PBS supplemented with 2% FBS at 25°C for 60 min in the dark. Appropriate antibody concentrations were determined previously by titration. FIX & PERM Cell Permeabilization Kit (Thermo Fisher, #GAS003) are intended for the fixation (Reagent A), permeabilization (Reagent B), and intracellular antibodies (Alexa Fluor 488 anti-SOX2 Antibody, BioLegend, #656110) of the cells. Samples were washed with PBS supplemented with 2% FBS and resuspended in 1× stabilizing fixative for use in flow cytometry.

A total of 6×10⁵ CMV-TCR-T cells/untransduced (UTD) T cells were co-cultured with an equivalent number of mixed U251-GFP-Luci-Vector cells (derived from a human glioma cell line and engineered with a green fluorescent protein and luciferase vector, previously constructed by our laboratory), were infected with CMV (Strain AD169, sourced from the Microbial Virus Strain Bank of Central South University). This co-culture took place at 37°C for 16 hours in Roswell Park Memorial Institute (RPMI) 1640, supplemented with L-glutamine (2 mM), antibiotics (penicillin 100 U/mL, streptomycin 100 µg/mL), and 10% FBS. Brefeldin A (10 µg/mL #420601, BioLegend) was added 4 hours before harvest. Following incubation, cells were washed twice with PBS containing 1% FBS, then stained with CD3 (BV421 Mouse Anti-Human CD3), CD8 (PerCP/Cyanine5.5 anti-human CD8a, BioLegend, #301032) and Zombie NIR Fixable Viability Dye Kit at 4°C for 30 min. After washing and fixation using the FIX & PERM Cell Permeabilization Kit (Thermo Fisher, #GAS003), cells were further stained with interferon (IFN)-γ (APC Mouse Anti-Human IFN-γ Antibody, BioLegend, #506510) for 45 min at 4°C. After two additional washes, cell analysis was conducted using an FACS flow cytometer, and FlowJo software was employed for data analysis.