Apc cy7 rat anti mouse cd19

For flow cytometry-based sorting of infected cells, mice were infected with 10⁴ PFU MHV68-H2bYFP, a phenotypically wild-type virus that expresses eYFP under control of the H2b promoter [44 (link)]. At 16 dpi, splenocytes were prepared and blocked as described above. Cells were then stained with APC rat anti-mouse CD4 at 1:200 (BD Biosciences, 553051), APC rat anti-mouse CD8α at 1:200 (BD Biosciences, 553035), APC rat anti-mouse CD14 at 1:100 (BD Biosciences, 560634), and APC-Cy7 rat anti-mouse CD19 at 1:200 (BD Biosciences, 557655). Infected B cells (CD4^-CD8^-CD14^-CD19⁺YFP⁺) and non-infected B cells (CD4^-CD8^-CD14^-CD19⁺YFP^-) were sorted using a BD FACSAria II flow cytometer (BD Biosciences). Sorted cells were immediately subjected to RNA extraction using an RNAqueous-Micro kit (Ambion, AM1931) prior to qRT-PCR analyses.

Fourteen weeks after transplantation of lentivirally transduced HSCs, splenic T cells (defined as CD3⁺ cells), B cells (defined as both CD19⁺ and MHCII⁺) or the remaining MHC Class II⁺ pool (defined as CD19^- and MHCII⁺ cells, i.e. non-B APCs) were sorted by FACS Synergy. Briefly, single-cell suspensions were prepared in PBS with 10% FCS. APC Anti-Mouse CD3e (eBioscience), APC-Cy7 Rat Anti-Mouse CD19 (BD Pharmingen, US) and eFlour450 Anti-mouse MHC Class II (I-A/I-E) (eBioscience), respectively, were added to the cell suspensions. Following 30 min incubation at 4°C, cells were sorted by FACS. 7AA-D (BD Pharmingen, US) were added just prior sorting for exclusion of dead cells. Purity and viability of isolated cells was around 99 (S1 Fig). Freshly isolated cell populations (1x10⁶ T cells, 2x10⁶ B cells and 3x10⁵ APC cells/mouse) were adoptively transferred into naïve syngeneic recipient mice (Fig 3B–3D). Recipient mice were administered the cells 2 days prior to CII immunization and evaluated for development of arthritis as described above.