Cd20 apc clone l27

A series of commercially available human monoclonal antibodies that cross-react with NHP mononuclear cells were used in flow cytometry analyses, as described previously [11 (link), 13 (link), 14 (link)]. Briefly, 100 μL of whole blood from each sample was added to each 12-mm × 75-mm polystyrene test tube (Falcon, Lincoln Park, NJ) containing panel of monoclonal antibodies CD3 Percp (clone SP-34), CD8 PE (clone SK1), CD16 FITC (clone 3G8) and CD20 APC (clone L27) (all from BD Biosciences, San Diego, CA) and incubated for 15 min at room temperature in the dark. Red blood cells were lysed with 1× FACS lysing solution (Becton Dickinson, San Diego, CA), diluted according to the manufacturer's instructions. The samples were washed thoroughly in 1× phosphate-buffered saline (PBS) by centrifugation; cell sediments were then suspended in 1% paraformaldehyde buffer (300 μL), and cells were acquired on a 4-color flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA). Lymphocytes that were gated on forward scatter versus side scatter dot plot were used to analyze CD3⁺, CD4⁺(CD3+CD8-),CD8⁺ (CD3+CD8+) T-CD16+ (NK cell), CD3+CD16+ (NKT) and CD20⁺ B-cell lymphocyte subsets with use of FlowJo software (Tree Star, Inc., Ashland, OR).

Peripheral blood mononuclear cells and subcortical white matter‐derived single cell fractions were isolated after rapid post‐mortem autopsies of NBB brain donors as described previously 56, 57. Donor and sample characteristics are listed in Table S7. Cells were stained with fixable viability dye eFluor 780 (Life Technologies) and the following antibodies: CD3 PE‐Cy5.5, clone SK7, (Invitrogen), CD20 APC, clone L27, CD25 FITC, clone 2A3, CD69 BV395, clone FN50, CD127 PE, clone HIL‐7R‐M21, (BD Biosciences), CD4 BV510, clone RPA‐T4, CD8a BV785, clone RPA‐T8, and FAS PE‐Cy7, clone DX2, (Biolegend), and analyzed on a Fortessa LSR^TM cell analyzer (BD Biosciences, San Jose, CA, USA). Data were analyzed with FlowJo software 10.5 (Tree Star, Ashland, OR, USA).