Anti bcl6 pe

For CD4⁺CD25⁺Foxp3⁺ T cells, splenocytes were isolated using standard methods. Nuclear stain for transcription factor FoxP3 was performed with the BD Biosciences (San Jose, CA) kit. Briefly, splenocytes (1×10⁶ per mouse) were stained with fluorescent conjugated antibodies, including anti-mouse CD4-pacific blue, anti-mouse CD25-PE and anti-mouse FoxP3-Alexa Fluor 647 according to staining protocol. For TfH cell staining, anti-CXCR5-BV421, anti-CD4-BV605, anti-PD-1-APC, anti-B220-FITC, and anti-Bcl6-PE were purchased from BD Bioscience (San Jose, CA). Fixable viability dye was purchased from eBioscience (San Diego, CA). For dendritic cell characterization, antiCD11b, anti-CD11c, and anti-CD64 were purchased from eBioscience (San Diego, CA). FACS analysis using LSR-II instrumentation from BD Biosciences (San Jose, CA) and the FCS Express software (DeNovo Software).

For cytokine staining, total splenoyctes were stimulated with PMA and Ionomycin for 4 h at 37°C. Brefeldin A (10μg/ml) was added after 2 h of stimulation. Following a surface stain, cells were fixed in 4% paraformaldehyde and permeablized in 0.1% saponin for 30 min at 4°C. Cytokines were then stained for 30 min at 4°C. Bim, Bcl-2, Ki67, Bcl-6, and T-bet were stained using the eBioscience Foxp3 staining buffer kit following manufacturer’s recommendations. Bim Ab was stained with a fluorescent Goat α-Rabbit Ab from Invitrogen. Host IA^b-NP_311-325-specific CD4 T cells were stained with the IA^b-NP_311-325-APC tetramer obtained from the NIH Tetramer Core Facility. All antibodies were obtained from eBioscience except anti-Bim (Cell Signaling) and anti-Bcl-6-PE (BD biosciences). Gating strategy includes gating on single cells, lymphocytes, and live cells distinguished by Invitrogen Live/Dead cell viability dye. Samples were run on LSRII instruments (BD biosciences) and analysis was done using Flowjo (Tree Star) analysis software.

For CD40L intracellular staining, sorted T cells were stimulated with PMA (100 ng/ml) and ionomycin (1 μg/ml) for an hour, fixed (BD CytoFix/CytoPerm™) and permeabilized (BD PERM/Wash™ solution). After permeabilization, cells were stained with anti-CD40L PE (24-31, Biolegend, 1:20 dilution). For IL-10 staining, total lymphocytes were stimulated with PMA (100 ng/ml) and ionomycin (1 μg/ml) for four hours and then for an additional two hours in the presence of Golgi plug (BD). Cells were stained for surface molecules prior to fixation. After fixation, cells were permeabilized and stained with anti-IL-10 Alexa Fluor 647 (JES3-9D7, Biolegend, 1:20 dilution). For BCL6, BLIMP-1, and Ki67 staining, total lymphocytes were stained for surface molecules prior to fixation. After fixation, cells were permeabilized and stained with anti-BCL6 PE (K112-91, BD, 1:50 dilution), anti-BLIMP-1 PE (6D3, BD, 1:50 dilution), or anti-Ki67 FITC (MOCP-21, BD, 1:100 dilution). FOXP3 staining was analogous to that of BCL6 except for changes in fixation and permeabilization (Biolegend FOXP3 FIX/PERM kit) and the use of anti-FOXP3 PE (206D, Biolegend, 1:10 dilution). Samples were loaded on LSR II (BD). Events were collected and analyzed with FlowJo software (TreeStar, CA).