Pacific blue cd150

Bone marrow cells were harvested from tibia and femurs of mice by gentle flushing with HBSS++ buffer [Hanks balanced salt solution (10–547F, Lonza) + HEPES (BP299–100, Fisher Scientific) + Fetal bovine serum (F2442, Sigma) + penicillin-streptomycin (15140–122, GIBCO)]. Samples were filtered through a 70 μM filter to obtain a single cell suspension and Lin⁻ enrichment was performed with lineage negative selection using the lineage cell depletion kit (130–090-858, Miltenyi). For isolation of LSK cells and LT-HSCs, Lin⁻ cells were incubated with biotin-labeled lineage antibody cocktail from BD containing a mixture of antibodies against CD3, CD11b, CD19, B220, Gr-1 and Ter119 (51–09082J, BD Pharmingen), followed by staining with streptavidin-PE secondary antibody (554061, BD). LSK cells were identified as Lin-Sca-1+c-kit+ using PE-Cy7-Sca (Clone D7, 558162, BD Biosciences) and APC-c-kit (Clone ACK2, 135108, BD Biosciences) antibodies; whereas LT-HSCs were recognized by being LSK and CD150+CD48- using PE-Cy7-Sca (Clone D7, 558162, BD Biosciences), APC-c-kit (Clone ACK2, 135108, BD Biosciences), Pacific Blue-CD150 (Clone TC15–12F12.2, 115924, Biolegend) and APC-Cy7-CD48 (Clone HM48–1, 47–0481-82, e-Bioscience). Cells were sorted using a BD FACSAria cell sorter.

BM cells were harvested from tibia and femurs of mice by gentle flushing with HBSS++ buffer [Hanks balanced salt solution (10-547F, Lonza) + HEPES (BP299-100, Fisher Scientific) + Fetal bovine serum (F2442, Sigma) + penicillin-streptomycin (15140-122, GIBCO)]. Samples were filtered through a 70 μM filter and Lin- enrichment was performed using the lineage cell depletion kit (130-090-858, Miltenyi). LT-HSCs were recognized by being LSK (Lin-Sca-1+c-Kit+) and CD150+CD48- using PE-Cy7-Sca (Clone D7, 558162, BD Biosciences), APC-c-Kit (Clone ACK2, 135108, BD Biosciences), Pacific Blue-CD150 (Clone TC15-12F12.2, 115924, Biolegend) and APC-Cy7-CD48 (Clone HM48-1, 47-0481-82, e-Bioscience).