Tnf α fitc mp6 xt22

The following fluorescence conjugated anti-mouse mAbs were purchased from eBioscience (San Diego, CA, USA): CD4-APC (L3T4), CD11c-APC and PE-Cy7 (N418), CD44-PE (IM7), CD86-PE (GL1), MHC Class II-FITC (M5/114.15.2), NK1.1-PE-cy7 (PK136), IgA-FITC (mA-6E1), IgM-APC (II/41) and TNF-α-FITC (MP6-XT22). B220-PerCP and APC-Cy7 (RA3-6B2), CD3ε-PerCP (145-2C11), CD8α-PerCP (53-6.7), CD11b-APC-Cy7 (M1/70), IFN-γ-PE (XMG1.2) and IL-12p40/p70-PE (C15.6) were purchased from BD Pharmingen (San Diego, CA, USA). PDCA-1-APC (JF05-1C2.4.1) was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

The retinal cells were stained with fluorescent dyes conjugated to antibodies as follows: CD206-FITC (C068C2; BioLegend, San Diego, CA, USA); 7-AAD (BD Pharmingen, San Jose, CA, USA); CD11b-APC-Cy7 (M1/70; BD Biosciences San Jose, CA, USA); f4/80-PE-Cy7 (BM8; BioLegend). After staining, the cells were washed twice with a FACS buffer (0.5% BSA in PBS buffer) and suspended in the FACS buffer for FACS analyses. Data were collected using a FACS Canto II flow cytometer (BD Biosciences) and analyzed by FCS Express software (De Novo Software, Los Angeles, CA, USA). The 7-AAD− CD11b+ f4/80+ cells were defined as retinal macrophages.
To investigate intracellular cytokine production, retinal cells were treated with a leukocyte activation cocktail with BD GolgiPlug^TM (BD Biosciences) for 4.5 h and with a BD Cytofix/Cytoperm Fixation/Permeabilization^TM kit (BD Biosciences) and then stained with the following antibodies: TNF-α-FITC (MP6-XT22; eBiosciences, San Diego, CA, USA); IL-10-PE (GK1.5; BioLegend); CD11b-APC-Cy7 (M1/70; BD Biosciences); f4/80-PE-Cy7 (BM8; BioLegend); and 7-AAD (BD Pharmingen, San Jose, CA, USA). The 7-AAD− CD11b+ f4/80+ cells were defined as retinal macrophages. Data were collected using a FACS Canto II flow cytometer (BD Biosciences) and analyzed by FCS Express software (De Novo Software).