Cd33 apc vio770

Animal studies were approved by the state animal research committee (LANUV, NRW, Germany) and all animals were cared for according to the guidelines set by the Federation of European Laboratory Animal Science Associations. Six- to 8-week-old female NOD.Cg-Prkdc^SCIDIl2rg^tm1Wjl/SzJ (NOD-SCID gamma; NSG) mice (Charles River Laboratories, Sulzfeld, Germany) were intravenously engrafted with 3.5 × 10⁶ MOLM-14 cells stably expressing a firefly luciferase-EGFP fusion protein (LucEG). Six days later, mice were intravenously injected with 3.5 × 10⁶ N3-, N4-, or CD8-hinged CD19 or CD33 CAR T cells. At days 6, 13, 20, 27, and 34, the persistence of MOLM-14 cells was assessed via luminescence imaging and PB analysis. For luminescence imaging, mice were intraperitoneally injected with D-luciferin (OZ Biosciences SAS, Marseilles, France) and after 5 min luminescence was measured in a Caliper IVIS Lumina II system (PerkinElmer LAS, Rodgau, Germany) with an exposure time of 15 s. PB was drawn from the tail vein, the erythrocytes lysed with BD Pharm Lyse (BD Biosciences), and the samples analyzed on a MACSQuant Analyzer X flow cytometer for EGFP, CD33, and CD45 expression for MOLM-14 cells and BFP, CAR (ΔNGFR), CD3, and CD45 expression for CAR T cells after staining with CD271-PE, CD3-PerCP-Vio700, CD45-APC, and CD33-APC-Vio770 (all from Miltenyi Biotec)