Cd56 pe clone hcd56

NK cell conversion was assessed by flow cytometry using CD45-Pacific Blue (clone HI30), CD56-PE (clone HCD56), CD3-APC700 (clone UCHT1), CD9-FITC (clone HI9a), and CD103-APC (clone Ber-ACT8), with Zombie NIR dye to assess viability (all from BioLegend, San Diego, California). Intracellular staining for VEGF-A protein was performed using the following antibodies: VEGF-A-APC (clone: 23410, R&D Systems) and VEGF-A (clone: EP1176Y, Abcam). For intracellular assays, cells were cultured for 3 days under various conditions with brefeldin A (1:1,000; BD Biosciences, Franklin Lakes, NJ) added for the final 6 h of the culture. Following staining for viability and surface expression of CD45, CD56, and CD3, samples were fixed, permeabilized, and stained for intracellular VEGF-A. Analysis was performed using FlowJo and cells were gated to only include live, CD45⁺, CD56+, CD3⁻ NK cells.

Human cells were washed with PBS and staining buffer, and then incubated with Human TruStain Fc receptor blocking solution (BioLegend, #422302) for 15 minutes. Cells were stained with the following fluorochrome-conjugated monoclonal antibodies: CD3-FITC (clone HIT3a, BioLegend), CD56-PE (clone HCD56, BioLegend), CD45-BV510 (clone HI30, BioLegend) and CD8-BV785 (clone RPA-T8, BioLegend). Live/dead staining was performed using Fixable Viability Dye 780 (eBioscience #65-0865-14). Data were acquired using a BD LSRFortessa flow cytometer (Becton Dickinson, San Jose, CA) and analyzed using FlowJo Software (Beckton Dickinson, San Jose, CA). Fluorescence-associated cell sorting (FACS) was performed using a BD inFlux cell sorter (Beckton Dickinson, San Jose, CA). Cells were sorted for blood and intra-tumoral NK cells (live CD45⁺CD3^-CD56⁺), blood and intra-tumoral T cells (live CD45⁺CD3⁺CD56^-), and tumor cells (live CD45^-). Following FACS isolation, cells were cryopreserved and stored in liquid nitrogen for subsequent RNA extraction and sequencing.