Fitc conjugated anti cd4 antibody

Non-specific staining was blocked by incubating cells with an anti-FcR antibody in 0.5% BSA at 37°C for 30 minutes. Cells were subsequently immunostained with the following antibodies: PE/cy5-conjugated anti-CD45, Alexa Fluor 488-conjugated anti-CD11c, APC-conjugated anti-CD86 and PE/cy7-conjugated anti-MHC-II (Biolegend) For TSP-1 staining, cells were incubated with primary TSP-1 antibody (abcam) at 4°C overnight, and stained with the goat anti-mouse IgG conjugated with Dylight 488 (abcam). To quantify IL-17-secreting CD4⁺ cells, single cell suspension was prepared from cervical LNs harvested from human serum albumin (HSA)-treated and recombinant TSP-1-treated DED mice on day 14. Cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Sigma-Aldrich Corp.) in the presence of GolgiStop (BD Biosciences), and subsequently stained with an FITC-conjugated anti-CD4 antibody (Biolegend). After fixation and permeabilization (buffers from eBioscience, San Diego, CA, USA), cells were stained with a PE-conjugated anti-IL-17A antibody (eBioscience). Appropriate isotype-matched control antibodies were used in all experiments. Cells were analyzed using the LSR II flow cytometer (BD Biosciences, San Jose, CA) and data were analyzed using FlowJo vX.0.7 software (FlowJo LLC., Ashland, OR, USA).