Apc conjugated anti human pd 1 antibody

Female 8-week-old NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA), and housed at the Kyoto Prefectural University of Medicine for more than a week before starting the experiment. Food and water were provided ad libitum. REH-FFLuc-GFP cells suspended in D-PBS were infused into mice via the tail vein. Six days later, RA⁺ CAR-T, RA⁻ CAR-T, or irrelevant CAR-T cells, which redirected the EPHB4 receptor (17 (link)) as a control, were infused via the tail vein, and tumor burdens were monitored using the IVIS Lumina Series III system (PerkinElmer, Inc.). The regions of interest on the displayed images were quantified in photons per second (ph/s) using Living Image v2 (PerkinElmer, Inc.) as described previously (17 (link)). Bone marrow (BM) cells were obtained by sequential BM aspiration from tibias at several time points. The BM cells were stained with a PE-conjugated anti-human CD3 antibody and APC-conjugated anti-human PD-1 antibody (BioLegend), and the long-term persistence of human T cells was evaluated by flow cytometry. The mice were euthanized at predefined endpoints, under conditions that met the euthanasia criteria given by the Center for Comparative Medicine at the Kyoto Prefectural University of Medicine.

The single-cell suspensions were generated in PBS buffer with 1% FBS and 0.1 mM EDTA. Cells were incubated with APC-conjugated anti-human PD-1 antibody (Clone EH12.2H7, Biolegend) for 30 min at 4 °C in the dark, then the cells were washed twice with PBS buffer containing 1% FBS and 0.1 mM EDTA. Flow cytometry data were acquired on a FACS flow cytometer (Beckman, Cytoflex) and analyzed with Flowjo 10.6.2 software (TreeStar).