Cd19 bv605 sj25c1

Mice were sacrificed after 14 weeks, and injected femur, and uninjected femur and tibiae were collected. Single cell suspension was prepared using standard flushing and cell dissociation techniques in 1 ml of IMDM. From that suspension, 100 μl of injected femur, 50 μl uninjected marrow (~1×10⁶ cells) were stained in a total volume of 200 μl staining buffer. Samples were not lysed with red blood cell lysis buffer as not to lyse human erythrocytes. Samples were stained with the 1:75 dilution the following antibodies: SytoxGreen live-dead stain (Thermo), CD33-PE (WM53; BD), CD45-APC (P67.6; BD), CD235a (11E4B-7–6; Coulter), CD19-BV605 (SJ25C1; BD), CD3-BV786 (SK7; BD), and CD66b-BV421 (G10F5; BD). All antibodies were confirmed as human-specific. Uninjected mouse marrow was used as a control for non-specific staining; CB mononuclear cells were used as a positive control. Compensation was performed using automated compensation with anti-mouse Ig^k and negative beads (BD Biosciences).

Transplanted human cord blood cells were harvested from mice 14 weeks post transplant. Cells harvested from 5 mice with equivalent engraftment for each condition were pooled and CD34⁺ cells isolated using the MACS selection column (Miltenyi, cat. #130–046-702). Cells were then stained with antibodies against human CD34-PE (581; BD), CD45RA-APC (HI100; BD), CD38-PC7 (HB7; BD), CD19-BV605 (SJ25C1; BD), CD10-BV786 (HI10a; BD), CD90-Biotin (5E10; BD) and analyzed on a BD FACS Aria III sorter.