Cd69 bv605 clone fn50

In total, 100 μL of EDTA-treated human whole blood was plated and pretreated with various concentrations of LY3541860 or isotype control antibodies for 30 minutes at 37°C. For induction of CD69 expression, 2.5 μg/mL of CpG (InVivogen) as final concentration was added in complete medium. No stimulation was designated as baseline. The plates were kept at 37°C and 5% CO₂ overnight. Then, cells were stained with the following antibodies for 30 minutes at 4°C: CD69 BV605 (clone FN50, BioLegend), CD20 PerCP Cy5.5 (clone 2H7, BD Pharmingen), CD19 APC (clone SJ25CI, BioLegend), and viability dye (eBioscience, 65-0865). FMO controls were used as gating controls. Cells were washed with staining buffer and run on the BD Fortessa X-20. Dead cells were excluded by viability dye, and at least 25,000–50,000 live cells were analyzed for each sample; results were analyzed using FlowJo software.

24-72 hours after co-incubation with beads, CAR-T cells were collected and stained with the following antibodies NGFR-FITC (clone ME20.4, Biolegend), Q8 (clone QBEND/10, ThermoFisher), CD45-AF700 (clone HI30, Biolegend), CD3-APC (clone SK7, Biolegend), CD4-PerCP (clone SK3, Biolegend), CD8-BV510 (clone SK1, Biolegend), PD-1-BV421 (clone EH12.2H7, Biolegend), CD69-BV605 (clone FN50, Biolegend), and HER2-PE-cy7 (clone 24D2, Biolegend). Live/dead discrimination was achieved using LIVE/DEAD fixable Near-IR dead cell stain kit (ThermoFisher). Flow cytometry results were analyzed using Kaluza software (Beckman Coulter).