Streptavidin pe texas red

The following antibodies were used: CD4-AlexaFluor 700, CXCR5-Biotin, ICOS-PECy7, Bcl-6-AlexaFluor 647, CD27-APCH7, CD45RA-PE, CD19-APC or V450, IgD-FITC, IL-21-AlexaFluor 647, IFN-γ-PECy7, IL-10-PE, Streptavidin-PE-TexasRed (BD Biosciences), CD38-PerCPeFluor710, IL-6-FITC, (Biolegend). Neutralizing antibodies specific for human IL-6 and IL-21R and isotype controls from R&D Systems.

Red blood cells were lysed with RBC lysis buffer (Invitrogen) and isolated splenocytes were stained with Live/Dead Aqua (ThermoFisher), or Fixable Viability Dye eF780 (ThermoFisher) for 20 min at 4°C in the absence of FBS, to differentiate live and dead cells. Cells were subsequently stained for surface markers (Supplemental table 1) in the presence of F_c block (αCD16/32, BioLegend) and 2% FBS. Cells expressing NP-specific BCRs were detected by staining with NP-PE (BioSearch). HEL peptide-loaded MHC II was detected using a mAb clone C3H4 (16 (link)) (kindly provided by Dr. Ron Germain, NIAID, Bethesda, MD), which was biotinylated and detected using streptavidin-PE-TexasRed (BD). After washing, cells were fixed in 1% paraformaldehyde (PFA) and stored at 4°C until analysis. Cells were analyzed on an LSRII flow cytometer (BD) and data were analyzed using FlowJo (v.10, Tree Star). See Figure 1A for gating strategies.

Monoclonal antibodies purchased from commercial sources are listed in Supplementary Table 1. Anti-mouse PDL-2, anti-mouse B7x, anti-mouse B7h and anti-mouse B7H3 antibodies were kindly provided by Xingxing Zang (Albert Einstein College of Medicine, Bronx, NY). A cocktail of antibodies against the lineage markers [CD5, CD45R (B220), CD11b, Gr-1 (Ly-6G/C), 7–4, and Ter-119 antibodies] was purchased from Miltenyi Biotec (San Diego, CA). Streptavidin-PE-Texas Red was from BD Biosciences (San Jose, CA). Foxp3/Transcription Factor Staining Buffer Set was from eBioscience (San Diego, CA). LTβR-Ig fusion protein was kindly provided by Biogen Idec MA Inc. (Cambridge, MA). Calcipotriol ointment was purchased from a local pharmacy.

We used flow cytometry to identify and enumerate blood basophils (CD49b⁺; IgE⁺), eosinophils (Siglec-F⁺; SSC^high), monocytes (Siglec-F⁻; CD11b⁺; Gr-1^low) and neutrophils (Siglec-F⁻; CD11b⁺; Gr-1^high), as well as peritoneal MCs (c-KIT⁺; IgE⁺). Briefly, blood cells were lysed by treatment with ACK buffer 2 times for 5 min. Cells were blocked with unconjugated anti–CD16/CD32 antibodies on ice for 5 min and then stained with a combination of the following antibodies on ice for 30 min: for blood leukocytes analysis, Siglec-F-PE (E50-2440; BD Biosciences), CD11b-eFluor450 (M1/70; eBioscience), CD49b-APC (DX5; eBioscience), IgE-biotin (23G3; eBioscience), and Gr-1-FITC (RB6-8C5; eBioscience). For peritoneal MCs, c-KIT-APC (ACK2; eBioscience), IgE-biotin (23G3; eBioscience). Cells were then incubated for 15 min with PE-Texas Red Streptavidin (BD Pharmingen). Data were acquired on LSRII and Accuri C6 (BD Biosciences) flow cytometers and analyzed with FlowJo software (TreeStar).

Spleen cells were first incubated with anti-murine Fcγ receptor II/III mAb, 2.4G2 for 10 min and then stained with saturating concentrations of Alexa Fluor 488-conjugated, APC-conjugated, biotin-conjugated, PE-conjugated, FITC-conjugated, PerCPCy5.5-conjugated, Alexa Fluor 647-conjugated and Pacific Blue-conjugated mAb against CD3, CD4, CD8, CD19, B220, H2-K^b, I-A^b, H-2K^d, and I-A^d, purchased from either BD Biosciences (San Jose, CA), BioLegend (San Diego, CA), eBioscience (San Diego, CA), or Invitrogen (Carlsbad, CA). Biotinylated primary mAb were detected using PE-Texas Red-streptavidin (BD Biosciences, San Jose, CA). Cells were fixed in 1% paraformaldehyde before reading.
Multi-color flow cytometric analyses were performed using a BD LSRII flow cytometer (BD Biosciences, San Jose, CA). Gating strategies: lymphocytes were gated by forward and side scatter and fluorescence data were collected for a minimum of 10,000 gated cells. Studies of donor T cells were performed on a minimum of 5,000 cells collected using a lymphocyte gate that was positive for CD4 or CD8 and either positive (host origin) or negative (donor origin) for MHC class I of the uninjected parent e.g. for B6→F1 mice donor cells are H-2K^d negative. B cells were gated as positive for B220 and either positive (host origin) or negative (donor origin) for MHC Class II of the uninjected parent.