Mcd45 apc cy7

Tail bleeds were analyzed on a Hemavet9500 (Drew Scientific). Engraftment was determined from flow cytometry of PB and BM preparations using a FACSCantoII instrument (BD) with analysis by FlowJo software. Our standard flow panel consists of antibodies to block mouse and human Fc IgG receptors (Miltenyi Biotech) as well as mCD45-APC/Cy7(BD), CD45-FITC (BD), CD3-PE/Cy7 (BD), CD19-VioBlue (Miltenyi Biotech), CD13-PE (BD), CD33-PE (BD), CD34-APC (BD), and CD56-v510 (BD). Leukemia percentage was determined by calculating the number of cells with positive staining for CD33 and CD45 (AML) or CD19 and CD45 (ALL) as a fraction of viable cells.

All mouse experiments were approved by the Austrian Ministry of Education, Science and Research (BMBWF-66.010/0056-V/3b/2019). 5 × 10⁵ CRISPR-modified HSPCs were intrafemorally transplanted into irradiated 8–12-week-old NSG mice. For competitive transplants, 1 × 10⁵ sort-purified HSPCs (1:1 mixture of CALR INS and WT cells) were intrahepatically transplanted into new-born NSGW41 mice. Human engraftment in murine bone marrow was evaluated after 8, 16, and 24 weeks via flow cytometry. Cells were stained with mTer119-BUV661, mCD45-APC-Cy7, hCD45-BB700, CD33-PE (BD Biosciences), CD19-SB600 (eBioscience, San Diego, CA, USA), CD41-PE-Cy7 (BioLegend) antibodies and SYTOX Red (Invitrogen, Carlsbad, CA, USA) and measured on a CytoFLEX LX flow cytometer (Beckman Coulter).