Anti b220 apc clone ra3 6b2

CH12F3 cells were transduced with VLP HA-Vpr or ΔVpr. 24 h later, transduced cells were stimulated to undergo IgM-to-IgA switching with 5 ng/mL mouse recombinant IL4 (Peprotech), 200 ng/mL anti-CD40 monoclonal antibody (clone HM40-3, eBiosciences) and 1 ng/mL hTGF-β (R&D Systems) for 3 days. After labeling cells with anti-B220-APC (clone RA3-6B2, eBiosciences) and anti-IgA-PE (Southern Biotechnology), CSR efficiencies (% of IgGA⁺ B220⁺ cells) were evaluated by flow cytometry. Primary mature mouse B-cells were transduced with Vpr-delivering VLPs or control VLPs, and stimulated 24 h later to undergo IgM-to-IgG1 switching with 40 μg/mL LPS (Salmonella typhimurium, R&D Systems) and 50 ng/mL IL-4 (PeproTech) for 3 days. After labeling with anti B220-APC (clone RA3-6B2, eBiosciences) and anti-IgG1-PE (clone A85-1, BD Biosciences), CSR efficiencies (% of IgG1⁺ B220⁺ cells) were evaluated by flow cytometry. Data were acquired on a BD LSRII Fortessa cytometer and analyzed with the BD FACSDiva 6.1.3 software. Cell proliferation was measured with standard MTT/formazan colorimetric assay at 550 nm [35 (link)].

The following anti-mouse antibodies were used: anti-CD16/CD32 Fc blocking antibody (clone 2.4G2) and anti–CD45-PE (clone 30-F11) (BD Biosciences); anti–CD24-FITC (clone M1/69), anti–SCA1-APC (clone D7) anti–Gr1-eFlour450 (clone RB6-8C5), anti–Ly6G-APC (clone 1A8-Ly6g), anti-F4/80 PerCP/Cy5 (clone BM8), anti–CD11b-PE-Cy7 (clone M1/70), anti–CD11c-APC-eFluor780 (clone N418), anti–CD4-FITC (clone 6K1.5), anti–CD8-PE (clone 53-6.7), and anti–B220-APC (clone RA3-6B2) (eBioscience); anti–CD61-Alexa Fluor 647 (clone 2C9.62[HMβ3-1]), anti–Ly6C FITC (clone HK1.4), anti–CD49b-eFluor450 (clone DX5), annexin V–APC (clone B217656) (BioLegend); and propidium iodide–PerCP (clone V13245) (Life Technologies).

The intracellular levels of cytokines and transcription factors were assessed using anti-CD4-eFluor450 (clone RM4-5), anti-C-X-C chemokine receptor type 5 (CXCR5)–peridinin chlorophyll protein complex (PerCP)–eFluor710 (clone SPRCL5), anti-B cell lymphoma 6 (Bcl-6)–APC (clone BCL-DWN), anti-IL-17–PE (clone eBio17B7), anti-forkhead box P3 (Foxp3)–PE (clone FJK-16 s), anti-B220–APC (clone RA3-6B2), anti-CD19–PerCP–Cy5.5 (clone eBio1D3), anti-IL-10–APC (clone JES5-16E3), anti-IL-17–fluorescein isothiocyanate (FITC) (clone eBio17B7; eBioscience), anti-T and B cell activation marker (GL-7)–FITC (clone GL7; BD Pharmingen), anti-CD1d–PE (clone 1B1), and anti-CD5–FITC (clone 53-7.3; eBioscience) antibodies. In brief, the cells were stimulated for 4 h with phorbol myristate acetate (25 ng/mL) and ionomycin (250 ng/mL) in the presence of GolgiStop. Next, the cells were incubated with fixable dye (eBioscience) for 30 min and permeabilized using Cytofix/Cytoperm solution (BD Pharmingen). Thereafter, the cells were reacted with the above-listed antibodies and analyzed using a CytoFLEX flow cytometer. Events were collected and analyzed with FlowJo software (Tree Star, Ashland, CA, USA).