Apc conjugated anti human cd45 antibody

The xenograft model was established with NRG/SRM3 mice obtained from the SBP Animal Facility. Mice were irradiated (2.5 Gy) and 2×10⁵ P31/Fujioka cells were then injected intravenously. At 9 days post-injection, engraftment of P31/Fujioka cells in NRG/SRM3 mice was confirmed by flow cytometric assessment of peripheral blood. (defined as ~1% human CD45+cells) and mice were randomized into two groups (n=5) to receive either PTC-596 (12.5 mg/kg) or vehicle (0.5% hydroxypropyl methylcellulose and 0.1% Tween 80 in distilled water) by oral gavage twice per week. Circulating leukemia cells were detected on day 18 by flow cytometry using a human-specific CD45 antibody (#404012, Biolegend, San Diego). Retro-orbital bleedings were performed 18 days after tail vein injections of P31 cells and 10 days after initial dosing of PTC-596 or vehicle control. Red blood cells were lysed using 1X PharmLyse Buffer (BD Biosciences), then washed with 1X PBS and stained with APC-conjugated anti-human CD45 antibody (#404012, Biolegend, San Diego) for 30 minutes in the dark, on ice. Cells were then washed again with 1X PBS and analyzed on the LSR Fortessa. Mice were sacrificed upon signs of morbidity resulting from leukemic engraftment (>10% weight loss, lethargy and ruffled fur).