Flow Cytometry Analysis of Immune Cells

Flow cytometry was performed as described previously [26] (link). Briefly, the single cell suspensions were prepared from thymus, spleen, and bone marrow of the mice at 7 days after receiving a single retrobulbar injection of 20 μL of 10 mM (56 μg) TPCA-1 in PBS or control vehicle, and passed through 70-μm nylon mesh (Fisher Scientific). After lysis of RBCs using ACK buffer (0.83% ammonium chloride, 0.1% potassium bicarbonate, 0.037% EDTA [pH 7.2]), Fc receptors were blocked with anti-CD16/32 antibody (Ab) before staining. To stain cell surface antigens, 10⁶ cells in 50 μL of staining buffer (1×PBS containing 3% BSA and 0.1% sodium azide) were incubated at 4°C for 30 minutes with fluorochrome-conjugated mAbs, and analyzed on a four-color BD FACSCalibur (BD Biosciences), the data was analyzed with CellQuestPro 5.1.1 software (BD Biosciences). Antibodies used for staining were as follows: rat anti-mouse CD16/32 blocking Ab (clone 2.4G2) was obtained from BD Biosciences (San Diego, CA); and rat anti-mouse TCR-β-FITC (H57-597), CD8a-PE (53-6.7), CD4-APC (GK1.5), B220-PE (RA3-6B2), IgD-APC (11-26), IgM-FITC (eB121-15F9), CD11b-APC (M1/70), Ly6C-PE (HK1.4), Ly6G-FITC (RB6-8C5), 7-amino-actinomycin D (7-AAD), and PE-conjugated rat IgG2a or IgG2b isotype Ab controls were purchased from eBiosciences (San Diego, CA).