Cd19 pacific blue clone sj25 c1

Fresh EDTA whole blood samples (500 μL) were stained with antibodies CD3-QD655 (clone S4.1, Invitrogen), CD4-QD605 (clone S3.5, Invitrogen), CD8-PECy7 (clone 3B5, BD Biosciences), HLA-DR-FITC (clone G46-6, BD Biosciences), and CD38-APC (clone HIT2, BD Biosciences). CD14-Pacific blue (clone TÜK4) and CD19-Pacific blue (clone SJ25-C1) from Invitrogen were used to exclude monocytes and B cells. Stained cells were acquired on an 18-color LSR II flow cytometer (BD Immunocytometry Systems) and analyzed using FlowJo software. The gating strategy (Supplementary Figure 2) was to gate on lymphocytes in the scatterplot, then singlets, then live CD3⁺ and CD14^– and CD19^– cells. From the CD3⁺ cell population, CD4⁺ and CD8⁺ cell populations were selected. Activation was measured as percentage of CD38⁺HLA-DR⁺CD8⁺ or CD38⁺HLA-DR⁺CD4⁺ T cells.

For profiling of ruxolitinib sensitivity of PBMC cell populations, PBMC from buffy coats were plated on V-bottom 96-well plates at 100,000 cells/well in 100 µl R10 supplemented with 2.5 ng/mL IL-2 in the presence of indicated concentrations of ruxolitinib or DMSO as a control, all conditions in triplicate. After 72 h incubation at 37 °C and 5% CO₂, cells were centrifuged, resuspended to 25 µl staining buffer (PBS + 0.5% BSA + 0.02% NaN₃) and stained with antibodies for CD56-FITC (clone NCAM16.2), CD4-PE-Cy7 (clone RPA-T4), CD3-APC (clone SK7), CD8-APC-H7 (clone SK1), CD14-V500 (clone M5E2) (all from BD Biosciences), and CD19-Pacific Blue (clone SJ25-C1, Invitrogen) for 15 min at RT. Cells were then washed with 100 µl staining buffer and resuspended to 25 µl Annexin V binding buffer with AnnexinV-PE and 7-AAD (both from BD Biosciences). Cells were acquired using an iQue Screener Plus flow cytometer (Intellicyt) and cell populations were gated as shown in Supplementary Figure 9 by first gating for viable cells based on AnnexinV/7-AAD negativity and then gating PBMC subpopulations. B cells were gated as CD19⁺ and monocytes as CD14⁺ cells. Counts of viable cells in each population per well were normalized to counts in DMSO-only wells (100% viability) and zero viable cells (0% viability).