Percp cy5.5 conjugated anti cd45 clone 30 f11

Ears of mice were separated in two sheets (ventral and dorsal) using forceps and enzymatically digested in RPMI 1640 medium containing 1 mg/ml collagenase (Sigma) and 50 ng/ml DNase (Sigma-Aldrich) for 60 min at 600 rpm and 37°C, and passed through a 70 μm cell strainer. Surface staining of cells was done by using APC or APC-Cy7 conjugated anti-CD45.2 (clone 104), APC, PerCP-Cy5.5 or APC-Cy7 conjugated anti-CD45.1 (clone A20), BV421 conjugated anti-F4/80 (clone BM8), Pe-Cy7, APC or APC-Fire conjugated anti-CD11c (clone N418), PE-Cy7 or APC-Cy7 conjugated anti-Ly6C (clone HK1.4), FITC conjugated anti-CCR2 (clone SA203G11), BV510 conjugated anti-MHC class II (IA/IE, clone M5/114.15.2), APC or APC-Cy7 conjugated anti-CD11b (clone M1/70), and PerCP-Cy5.5 conjugated anti CD45 (clone 30-F11), which were all purchased from BioLegend. Samples were Fc-blocked using anti-CD16/32 antibody (clone 93) (BioLegend) before antibody staining. Analysis was performed with a Fortessa or FACS ARIA III (BD Biosciences) using 405, 488, 561, and 633 nm lasers:. Photoconverted or non-photoconverted mKikume fluorescence was read out at 561 nm excitation and 610/20 nm emission, or 488 nm excitation and 530/30 nm emission, respectively. An autofluorescence signal was recorded at 488 excitation and 695/40 nm emission. Data were analyzed by using the FlowJo X software (FlowJo, LLC).

Anti-CD3 (clone 145-2C11), anti-CD28 (clone 37.51), anti-IL-4 (clone 11B11), and anti-IFN-γ (clone XMG1.2) antibodies for T-cell activation and differentiation; anti-FasL (clone MFL3 and MFL4) blocking antibodies; staining antibodies Alexa Fluor 488-conjugated anti-CD4 (clone GK1.5), PE-conjugated anti-CD8 (clone 53-6.7), -TCRβ (clone H57–597), -Foxp3 (clone 150D), -CD69 (clone H1.2F3), PerCP/Cy5.5-conjugated anti-CD45 (clone 30-F11), Alexa Fluor 647-conjugated anti-IFN-γ (clone XMG1.2), -CD3 (clone 145-2C11), APC-conjugated -CD25 (clone 3C7), FITC-conjugated Annexin V, 7-AAD, biotin-conjugated anti-FasL (clone MFL3), and APC-conjugated streptavidin were all purchased from Biolegend, San Diego, CA, USA. Alexa Fluor 647-conjugated goat anti-human IgG (H+L) for T-cell binding assay was obtained from Thermo Fisher Scientific, Waltham, MA, USA. Fc Block was from Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany, and FACS staining buffer (BSA) was from BD Pharmigen, San Diego, CA, USA. Anti-human Fd-capturing antibody (clone HP6045) and horseradish peroxidase (HRP)-conjugated goat anti-human kappa light chain detecting antibody for ELISA were from Sigma-Aldrich, St. Louis, MO, USA and Bethyl Laboratories, Montgomery, TX, USA, respectively.