Alexa fluor 750 anti cd45

Freshly isolated PBMCs or TILs (1 × 10⁶) were washed and incubated with fluorophore-conjugated monoclonal antibodies including Alexa Fluor 750-anti-CD45, Alexa Fluor 700-anti-CD3, BV421-anti-TCRγδ, FITC-anti-Vδ1, and PE-anti-Vδ2 (all from Biolegend, San Jose, CA, USA) for 20 min at room temperature in the dark. For intracellular cytokine detection, PBMCs or TILs (1 × 10⁶) were resuspended in RPMI 1640 medium supplemented with 10% FBS (Gibco) and stimulated with phorbol-12-myristate 13-acetate (50 ng/ml), ionomycin (1 μg/ml), and brefeldin (1 μg/ml) (all from Biogems, Rocky Hill, NJ, USA) for 5 h in 5% CO₂ atmosphere at 37 °C. Cells were then washed, fixed, permeabilized, and stained with APC-anti-IL-17A, and APC/cy7-anti-IFN-γ (Biolegend, San Jose, CA, USA) according to the manufacturer’s protocol. Fluorescence data were collected on a FACS Aria II (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo software (Tree Star, Ashland, OR, US).

γδ T cells (1 × 10⁵) sorted from healthy PB were cultured with OC tissues supernatants, BOT tissues supernatants, and RPMI 1640 control medium in 24-well plates in 37 °C at 5% CO₂. After 5 days, γδ T cells were harvested and stained with Alexa Fluor 750-anti-CD45, Alexa Fluor 700-anti-CD3, BV421-anti-TCRγδ, FITC-anti-Vδ1, and PE-anti-Vδ2 (all from Biolegend, San Jose, CA, USA) to analysis the levels of Vδ1 T cells and Vδ2 T cells by flow cytometry.

γδ T cells were sorted from PBMCs or TILs by fluorescence-activated cell sorting (FACS), following staining with Alexa Fluor 750-anti-CD45, Alexa Fluor 700-anti-CD3, andBV421-anti-TCRγδ (all from Biolegend, San Jose, CA, USA), using a FACS Aria II cell sorter; the purity of the sorted cells were greater than 95%.
The human OC cell line SKOV3 was purchased from ATCC (American Type Culture Collection, Manassas, VA, USA), and cultured in McCoy’s 5A medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS at 37 °C in 5% CO₂.