Cd45 pacific blue clone hi30

Whole blood was stained with antibodies and fixed with Optilyse C (Beckman Coulter, Krefeld, Germany) according to the manufacturer's instructions. Data were acquired on a Gallios flow cytometer (Beckman Coulter) and analyzed using FlowJo software (version 10; Treestar, Ashland, OR, USA). Antibodies were: CD4 PerCP (clone SK3), CXCR5 FITC (clone RF8B2) (all from BD Biosciences, Heidelberg, Germany), CD3 PE-Cy7 (clone UCHT1, Beckman Coulter), CD45 Pacific Blue (clone HI30), CD45RA APC-Cy7 (clone HI100), CCR6 BV605 (clone G034E3) (all from BioLegend, San Diego, CA, USA), CXCR3 PE (clone WM59, R&D Systems, Minneapolis, MN, USA).

Flow cytometry was performed using a NovoCyte cytometer (ACEA, Agilent, Santa Clara, CA, USA). The following antibodies were purchased from Biolegend (San Diego, CA, USA): CD3-FITC (clone OKT3, RRID: AB_571907), CD3-APC(clone OKT3, RRID: AB_1937212), CD107a-PE-Cy7(clone H4A3, RRID: AB_11147955), CD70-PE-Cy7 (clone 113-16, RRID: AB_2687254), PE anti-Streptavidin (clone 3A20.2, RRID: AB_2571915), CD34-PE (clone 581, RRID: AB_1731862), CD33-FITC (clone HIM3-4, RRID: AB_314344), CD123-APC (clone 6H6, RRID: AB_439779), CD13-PerCP/Cyanine5.5 (clone WM15, RRID: AB_2561921), CD117-PE/Dazzle™594 (clone 2B8, RRID: AB_2564055), CD38-Alexa Fluor^®700 (clone HB-7, RRID: AB_2566424), CD45-Pacific blue (clone HI-30, RRID: AB_493655). Biotinlyted CD70 protein (cat#CDL-H82D7, AcroBiosystems) was purchased from AcroBiosystems (Beijing, China), anti-mouse FMC63 (clone R19M, RRID: AB_2857947, BioSwan Laboratories) antibody was purchased from BioSwan Laboratories (Shanghai, China).

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.