Anti thy 1.2 antibody

Bone marrow cells from female C57BL/6 mice were removed aseptically from femurs and tibias by flushing with ice-cold PBS. After red blood cell lysis, T cells were depleted from bone marrow cells by incubation with anti-Thy 1.2 antibody (BioLegend) for 30 min at 4°C, followed by another incubation with Low-TOX-M rabbit complement (Cedarlane) for 40 min at 37°C. Splenic T cells were purified by positive selection using anti-CD5 antibody-conjugated magnetic beads (Miltenyi, Auburn, CA). TCD-BM cells (5 × 10⁶) were washed and resuspended in 100 μL PBS and injected into lethally irradiated recipient female B6D2F1 mice through the lateral tail vein. To induce GVHD, 1 × 10⁶ splenic T cells were injected together with 5 × 10⁶ TCD-BM cells into recipient mice. B6D2F1 mice received twice total body irradiation (5.5 Gy each) with a 3 h interval between each irradiation to minimize the gastrointestinal toxicity.

Fluorescence-activated cell sorting (FACS) was done as previously described.¹⁸ (link) Briefly, mouse whole retinas at P3 were isolated and incubated with 0.25% (wt/vol) trypsin in PBS for 15 minutes at 37°C. Retinas were resuspended with 2% (wt/vol) BSA in PBS and incubated with anti-Cd73 antibody (BD, 561545) and anti-Thy1.2 antibody (BioLegend, 105307) for 30 minutes on ice. Cells were stained with propidium iodide to exclude dead cells. Thy1.2-single-positive and Cd73-single-positive fractions were collected using FACSAria II (BD).