Dl101

Single-cell suspensions from rhesus macaque tissues were used as input into the LEGENDScreen assay (Biolegend). Cell suspensions were incubated with Fc block (Human TruStain FcX, Biolegend) prior to labeling with fluorescently conjugated antibodies. To enable assessment of assay markers on cell derived from individual tissues, cells from bone marrow, lymph node, and PBMC were each labeled with anti-NHP CD45 (clone D058-1283, BD Biosciences) conjugated to a unique fluorescent tag to enable sample multiplexing and unmixing during analysis. Following labeling with CD45, samples were washed 2× with FACS buffer before being stained with antibodies targeting CD3 (SP34-2, BD Biosciences), CD20 (2H7, Biolegend), and CD138 (DL101, Biolegend). 1 × 10⁶ cells were used as input into each well of the LEGENDScreen assay. Samples were acquired on a Symphony A5 (BD Biosciences) using FACSDiva (BD Biosciences).

Single-cell suspensions isolated from whole rhesus macaque bone marrow or human bone marrow aspirates were prepared for cell sorting by first incubating the cells with Fc block (Human TruStain FcX, Biolegend). Following Fc block, cells were washed 1× with DPBS before being stained with a cocktail of fluorescently conjugated antibodies. For rhesus samples, cell suspensions were stained with antibodies targeting CD102 (CBR-IC2/2, Biolegend), CD20 (2H7, Biolegend), CD3 (SP34-2, BD Biosciences), CD64 (10.1, Biolegend), CD11b (ICRF44, Biolegend), CD45 (D058-1283, BD), and CD31 (WM59, BD Biosciences). For human samples, cells suspensions were stained with antibodies targeting CD102 (CBR-IC2/2, Biolegend), CD20 (2H7, Biolegend), CD45 (HI30, Biolegend), CD31 (ICRF44, Biolegend), and CD138 (DL101, Biolegend). Following staining, samples were washed 2× with DPBS before being resuspended in cRPMI. CD45 + cells were further enriched from rhesus samples by magnetic enrichment using anti-NHP CD45 microbeads (Miltenyi Biotech). Cell sorting was performed on a Sony SH800 instrument (Sony) using 100um microfluidic sorting chips. Cells were sorted into low-volume fetal bovine serum to maintain higher cell concentrations before being used in downstream applications.