Bv421 conjugated anti mouse cd11c

20 kDa 4-arm PEG-azide was purchased from JenKem Technology USA (Beijing, China). Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) and sodium ascorbate (NaAsc) were purchased from Sigma-Aldrich (St. Louis, MO). Copper (II) sulfate pentahydrate (CuSO₄ • 5 H₂O) was purchased from Acros Organics (Geel, Belgium). Alkyne functionalized peptide bearing an N-terminal 4-pentynoic acid (homopropargyl, hp) modification, hpPLP_139–151 (hp-HSLGKWLGHPDKF-OH), was obtained from Biomatik, USA, LLC (Wilmington, DE). All reagents were used as received without further purification. For in vitro cell assays and in vivo studies, female 4–6-week-old SJL/J (H-2) mice were purchased from Envigo Laboratories (Indianapolis, IN). For EAE induction, incomplete Freund’s adjuvant (IFA) and heat-killed mycobacterium tuberculosis H37RA were purchased from Difco (Sparks, MD). Additionally, pertussis toxin was purchased from List Biological Laboratories (Campbell, CA). For use in flow cytometry, TruStain fcX (anti-mouse CD16/32), R-phycoerythrin (PE)/Cy7-conjugated anti-mouse CD3, PE-conjugated anti-mouse CD86, FITC-conjugated anti-mouse CD80, AlexaFluor647-conjugated anti-mouse CD19, and BV421-conjugated anti-mouse CD11c were purchased from BioLegend (San Diego, CA).

Lung cells were resuspended in 100 μl of staining buffer (PBS containing 2% fetal calf serum and 0.02% sodium azide) and incubated with anti-mouse CD16/CD32 (1:100; Biolegend, San Diego, CA) for 10 min at 4°C to block nonspecific binding. The cells were then incubated with FITC-conjugated anti-mouse CD11b (1:100, Biolegend), PE-conjugated anti-mouse Ly6C (1:100, Biolegend), BV-421 conjugated anti-mouse CD11c (1:100, Biolegend), AF 700-conjugated anti-mouse CD45 (1:100, Biolegend) and AF 647-conjugated anti-mouse Ly6G (1:100, Biolegend) antibodies for 30 min at 4°C followed by 30 min incubation with eFluor 780-conjugated fixable viability dye (1:100; eBioscience, San Diego, CA). Cells were then washed with PBS, fixed in 3% paraformaldehyde, and analyzed using a Beckman Coulter Gallios flow cytometer (Brea, CA). Data were analyzed using Beckman Coulter Kaluza software (version 1.2) as previously described (Sunil et al., 2015 (link)).