Cd20 percp cy5.5 clone 2h7

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.

Memory human B cells were isolated from healthy donor PBMCs using memory B cell isolation kit (Miltenyi Biotec, 130-093-546). Human primary memory B cells were resuspended at 1 × 10⁶ cells/mL and cultured at 37°C. Cells were pretreated with varying concentrations of either isotype control or LY3541860 as indicated for 30 minutes and stimulated with 50 ng/mL anti-CD40, 200 ng/mL BAFF, 1 ng/mL IL-2, and 100 ng/mL IL-21 (all from R&D Systems) for 5 days. Cells were washed and stained with the following combination of fluorochrome-conjugated antibodies — CD38 PE (clone HB-7), CD3 FITC (clone HIT3a), CD19 APC (clone SJ25CI, all from BioLegend), CD20 PerCP-Cy5.5 (clone 2H7, BD Pharmingen), and fixable viability dye eFluor 780 (65-0865, eBioscience) — in staining buffer for 30 minutes at 4°C to identify differentiation of memory B cells into plasmablasts. Samples were acquired on a BD Fortessa X-20, and results were analyzed using FlowJo software. Plasmablasts were defined as CD38^brightCD20^lo B cells.

Human primary B cells were plated at 1 × 10⁶cells/mL. Cells were treated with indicated concentrations of LY3541860 or isotype control for 24 hours at 37°C and 5% CO₂. B cell apoptosis was measured by flow cytometry using annexin V staining (Invitrogen) in combination with fixable viability dye. Cells were stained with the appropriate combination of fluorochrome-conjugated antibodies for 30 minutes at 4°C to identify B cell surface markers: CD19 APC (clone SJ25CI, BioLegend), CD20 PerCP-Cy5.5 (clone 2H7, BD Pharmingen), annexin V (Invitrogen), and fixable viability dye eFluor 780 (eBioscience, 65-0865). Apoptotic B cells were defined as live/annexin V⁺/CD20⁺. Samples were acquired on a BD Fortessa X-20 and results were analyzed using FlowJo software.