Cd19 apc sj25c1

For isolation of cells for scRNAseq, freshly isolated PBMC were FC block- treated (2 ul FC blocking reagent human (Miltenyi Biotech) and 48 ul staining buffer (1% BSA in PBS, filtered through a 40 um filter)/1x10⁶ cells) and incubated 10’ on ice. Cells were washed and stained in 50 ul final/1x10⁶ cells of antibody cocktail (CD3-APC (HIT3α, BD), CD19-APC (SJ25C1, BD), CD14-PE-Cy7 (M5E2, BD), CD56-PE-Cy7 (B159, BD), HLA-DR-APC-H7 (B159, BD), CD11c-AF700 (3.9, eBioscience), CD123-BV650 (6H6, Biolegends), CD16-BV605 (3G8, Biolegends)). Life- dead staining with 7AAD (BD) was done shortly before sorting on a FACS (ARIAII, BD) with the 100 uM nozzle using gating strategy shown in Supplemental Figure 1. For isolation of cells for qPCR, the same panel was used including also EMP1-FITC (Biozol) (Supplemental Figure 4A).

Frozen PBMC samples from pre-MP (n = 5), post-MP (n = 2) and males (n = 2), collected at 4 different time-points (as described above) were prepared for cell storing using FACS. The samples were thawed in a 37°C water bath and diluted with ice-cold PBS followed by 2 washing steps with ice-cold PBS by centrifugation (300 x g, 5 min) at 4°C. The cell pellet was resuspended in 200 μL ice-cold PBS. The cell suspension was incubated in darkness with respective antibodies (CD3 PE-Cy 7, Cat No 341111; CD4 PerCP-Cy 5.5, Cat No 332772; CD8 APC-H7 RUO, Cat No 641400; CD56 PE (MY31), Cat No 345810; CD19 APC (SJ25C1), Cat No 345791, all from BD Biosciences (San Jose, CA, USA), and CD14 [DakoAgilent, Santa Clara, CA, USA)], for 15 minutes and diluted with 2 mL PBS before centrifugation (600 x g, 5 min) at 4°C. Unbound antibodies (supernatant) were discarded and the cell pellet was resuspended in 400 μL PBS followed by cell sorting using FACSAria (BD Biosciences). At least 30’000 cells were collected from each category before storing at -80°C.