Apc conjugated anti cd86 gl 1

OVA_323–339 peptide was synthesized by the Emory University Microchemical Core Facility. Ovalbumin was from Worthington Biochemical Corporation and hen egg lysozyme was from Sigma-Aldrich. Some reagents were purchased: mouse IFN-γ (PeproTech), and lisinopril (Sigma). The PE-conjugated anti-CD69, PE-conjugated anti-CD80 (16–10A1), APC-conjugated anti-CD3ε, APC-conjugated anti-CD86 (GL-1) and the ELISA kits for IL-2, IFN-γ and IL-17A were all purchased from eBioscience. Alexa Fluor 488-conjugated Ovalbumin was from ThermoFisher. AF6–120.1 (anti-I-Ab),1G11 (anti-EEA1) and eBioH4A3 (anti-LAMP-1) were from eBioscience. A rabbit antiserum that recognizes mouse ACE was previously described (13 (link)). The FITC-conjugated mouse TCR Vβ Screening Panel was from BD Pharmingen. Flow cytometry stained samples were analyzed on CyAn ADP (Beckman Coulter) and data were analyzed with FlowJo software.

Single cell suspensions from mouse pancreas, spleen, and blood were prepared as previously described (Lee et al., 2016 (link)). Cells were stained in PBS/0.5% FBS/2 mM EDTA with the following fluorochrome-conjugated antibodies: V450-conjugated anti-CD3 (500A2, #560801, 1:100), APC-Cy7-conjugated anti-CD11b (M1/70, #561039, 1:50), V450-conjugated anti-CD11c (HL3, #560521, 1:100), APC-conjugated anti-CD19 (1D3, #550992, 1:200), PE-Cy7-conjugated anti-CD45 (30-F11, #552848, 1:100), V500-conjugated anti-CD45 (30-F11, #561487, 1:100), V450-conjugated anti-Gr1 (RB6–8C5, #560454, 1:200) (from BD Biosciences); APC-conjugated anti-CD86 (GL1, #17–0862, 1:100), PE-conjugated anti-F4/80 (BM8, #12–4801, 1:100) (from eBioscience); FITC-conjugated anti-CD206 (MR5D3, #MCA2235FT, 1:50) (from AbD Serotec); PE-Cy7-conjugated anti-MHCII (M5/114.15.2, #107629, 1:100) (from BioLegend). The viability marker 7-aminoactinomycin D (7-AAD) was purchased from BD Biosciences. Flow cytometry was performed on a FACSCanto flow cytometer (BD Biosciences), and data was analyzed using FlowJo software.