Pe cy7 anti cd20 2h7

PBMCs obtained following the 2^nd boost immunization were stained with 5-OP-RU tetramer conjugated with PE (NIH Tetramer Core Facility) for 30 minutes, followed by Alexa700 anti-CD3 (SP34-2), APC anti-CD4 (L200), FITC anti-CD8 (RPA-T8), and PE-Cy7 anti-CD20 (2H7) (BD Biosciences). Aqua Live/Dead viability dye was used to exclude dead cells. After staining, cells were washed, passed through a 40-mm cell strainer, and sorted on an Astrios EQ flow cytometer. Two groups of live cells were sorted (CD3⁺CD4⁻CD8⁺MR1⁺, and CD3⁺CD4⁻CD8⁺MR1⁻) to a purity of 95%. Live B cells were sorted using PE-Cy7 anti-CD20 (2H7) (BD Biosciences) from pre-vaccinated animals to a purity of 95%.

To allow identification of monocytic myeloid cells, PBMCs (5–10 × 106 cells) were stained with PE- Cy7 anti-CD20 (2H7; 560735, BD Biosciences), PE-Cy7 anti-CD3 (SP34-2; 563916, BD Biosciences), APC anti-CD14 (M5E2; 561390, BD Biosciences), BV786 anti-NHP-CD45 (D058-1283; 563861, BD Biosciences), HLA-DR-APC-Cy7 (L243; 307618, BioLegend), BV421 anti-CD192 (CCR2) (48607; 564067, BD Biosciences), FITC anti-CD16 (3G8; 555406, BD Biosciences) and PE-CF594 anti-CD184 (CXCR4) (12G5; 562389, BD Biosciences), as well as Aqua LIVE/DEAD kit (L34966, Invitrogen) to dismiss dead cells. CD45⁺Lin⁻ (CD3 and CD20) was considered as a signature of myeloid cell populations. Monocyte populations were further recognized and sub-categorized according to the expression of CD14 and CD16, where classical monocytes were identified as Lin⁻CD45⁺CD14⁺CD16⁻HLA-DR⁺, intermediate as Lin⁻CD45⁺CD14⁺CD16⁺HLA-DR⁺ and non-classical as Lin⁻CD45⁺CD14⁻CD16-HLA-DR⁺. Data are shown as frequency of CD45 cells or as frequency of HLA-DR cells. Acquisition was then done on an LSRII (BD Biosciences), and marker expression was examined in real-time using the software FACSDiva (BD Biosciences). Data were analysed more in detail with FlowJo version 10.1 (TreeStar, Inc.).