Cd45.1 fitc

Three week old CD45.2+ donor mice (wildtype, mirn23a^−/−mirn23b^f/f, and mirn23a^−/−mirn23b^f/f:MX1-CRE) were injected with pIpC as described above. Donor mice received a total of 6 injections over 10 days. Donor mice were sacrificed 2 weeks after the last injection. Femurs and tibias were removed for isolation of bone marrow. Whole marrow was pooled according to genotype. Red cells were removed by Ammonium-Chloride-Potassium (ACK) cell lysis buffer. Nucleated cells were mixed at 1:1 ratios (WT: WT CD45.1; mirn23a knockout:CD45.1; and mirn23a/23b double knockout:CD45.1), spun down, and re-suspended in ice cold PBS to a final concentration of 10⁷ cells/ml. Recipient mice were lethally X-ray irradiated (RS 2000 Biological Research Irradiator) 5–6 hours before transplant. A dose of 100ul cell suspension (10⁶ cells) was retroorbitally injected. Reconstitution was monitored by analysis of peripheral blood. Mice were cheek bled at 6 and 12 weeks post-transplant. Blood was collected in Eppendorf tubes coated with EDTA (0.5M). Red blood cells were removed by ACK lysis. Nucleated peripheral blood was stained for flow cytometry with CD45.1 FITC, CD45.2 PE, B220 APC, CD11b APC-Cy7 antibody conjugates obtained from BioLegend (San Diego, CA, USA).