Live dead fixable violet dead cells stain kit

To assess the effects of PN on T cell subsets, 1000 µL of PBMCs (1 × 10⁶) seeded into each well of a 24-well plate were treated with either the IC₅₀ concentration (325.3 μg/mL) of PN or 5 μg/mL phytohemagglutinin (PHA) or the combination of PN and PHA or left untreated and incubated for 24, 48, and 72 h at 37°C in a humidified atmosphere containing 5% CO₂. After incubation of treatment, the cells were centrifuged at 1500 rpm for 10 min to pellet the cells, and the cell culture supernatants were stored at −80°C for cytokine quantification. PBMCs were washed with sterile PBS supplemented with 2% FCS and then stained for 45 min at room temperature with an antibody cocktail containing CD3-APC-H7, CD4-PE-CY5.5, CD8-BV711, CD3-APC-H7, HLA-DR-A700, CD38-PE-Cy-7, CCR5 APC, CCR6 BV605, CD14-Pacific Blue (monocyte exclusion), CD19-Pacific Blue (NK cells exclusion/B cells exclusion), and viability markers (Live/dead ™ Fixable Violet Dead Cells Stain Kit, Invitrogen (Thermo-Fisher), Massachusetts, USA). The cells were washed with Perm/Wash buffer and acquired on the BD LSR Fortessa (BD Biosciences, Franklin Lakes, NJ, USA). At least 500 000 events were acquired from each sample. Data analysis was performed using FlowJo v10.4.1 software (Tree Star, Ashland, OR USA), according to the gating strategy (Figure 1).

On day 1, target cells (GCT cell line and OVCAR-5) were stained with CellTrace CFSE (Invitrogen) following manufacturer’s recommendation and plated in a 24-well plate at 1 × 10⁵/well. The day afterward, increasing ratios of TILs were added to the wells, and the plate was incubated overnight. On day 3, cells were collected and stained with LIVE/DEAD fixable violet dead cells stain kit (Invitrogen, L34964) and with anti-CD3 (eBioscience; clone 17A2, 46-0032-82). Cells were analyzed on a FACSCanto flow cytometer using FlowJo software.