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MB Sample ID: SA323642
| Local Sample ID: | HSCa_31 |
| Subject ID: | SU003096 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
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Collection:
| Collection ID: | CO003089 |
| Collection Summary: | Bones were rapidly dissected and stored on ice in Ca2+- and Mg2+-free HBSS (ThermoFisher) plus 2% heat-inactivated fetal bovine serum (Gibco). Bone marrow cells were flushed out from the bone and then dissociated to single-cell suspension by gently passing through the needle then filtering through a 70-μm nylon mesh. The following antibodies were used to isolate hematopoietic cells: TER-119-FITC, CD3-FITC, CD5-FITC, CD8-FITC, B220-FITC, Gr-1-FITC, TER-119-APC780, CD3e-APC780, CD5-APC780, CD8-APC780, B220-APC780, Gr-1-APC780, TER-119-biotin, CD3-biotin, B220-biotin, Gr-1-biotin, c-kit-biotin, TER-119-PerCP/Cy5.5, Sca-1-PerCP/Cy5.5, MAC-1-APC, CD48-APC, CD16/32-APC, CD135-APC, CD127-PE, CD34-PE and CD3-PE, CD150-PE. FITC streptavidin, APC/Cy7 streptavidin and/or APC-R700 streptavidin were used for biotin-labeled antibodies. All reagents were acquired from BD Biosciences, eBiosciences or BioLegend. For isolation of HSPCs, cells were incubated with c-kit-biotin and paramagnetic microbeads, and then passed through an autoMACS magnetic separator. For isolation of CLPs, lineage was stained with Ter119-biotin, CD3-biotin, B220-biotin and Gr-1-biotin and paramagnetic microbeads. Then an autoMACS magnetic separator was used to enrich lineage- populations. To minimize metabolic changes, antibody-stained cells were fixed with 2% PFA (Sigma) for 15 minutes on ice. Flow cytometric analysis was performed with a BD LSRFortessa cytometer. To measure ROS levels of HSCs, antibody-stained cells were incubated with 5μm DCFDA for 15 minutes at 37°C before flow cytometric analysis. For metabolic detection, cells were sorted with a FACSAria SORP cytometer into 384-well plates containing 2.5-μl 80% methanol (Sigma) per well, and then centrifuged at 1500g for 5 mins at 4°C. Plates were sunk in liquid nitrogen for 10 mins to lyse cells and kept at -80°C before MS analysis. |
| Sample Type: | Stem cells |
