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MB Sample ID: SA323780
| Local Sample ID: | HSCd_1 |
| Subject ID: | SU003096 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003096 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| HSCd_1 | SA323780 | FL038667 | HSCd | Celltype |
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 |
Treatment:
| Treatment ID: | TR003105 |
| Treatment Summary: | 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. |
Sample Preparation:
| Sampleprep ID: | SP003102 |
| Sampleprep Summary: | 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. |
Combined analysis:
| Analysis ID | AN004902 |
|---|---|
| Analysis type | MS |
| Chromatography type | None (Direct infusion) |
| Chromatography system | none |
| Column | none |
| MS Type | MALDI |
| MS instrument type | MALDI-TOF-MS |
| MS instrument name | Bruker Autoflex MALDI-TOF (/TOF)-MS |
| Ion Mode | POSITIVE |
| Units | intensity |
Chromatography:
| Chromatography ID: | CH003697 |
| Chromatography Summary: | None |
| Instrument Name: | none |
| Column Name: | none |
| Column Temperature: | none |
| Flow Gradient: | none |
| Flow Rate: | none |
| Solvent A: | none |
| Solvent B: | none |
| Chromatography Type: | None (Direct infusion) |
MS:
| MS ID: | MS004645 |
| Analysis ID: | AN004902 |
| Instrument Name: | Bruker Autoflex MALDI-TOF (/TOF)-MS |
| Instrument Type: | MALDI-TOF-MS |
| MS Type: | MALDI |
| MS Comments: | MS acquisition Comments: MS experiments were conducted on the Autoflex MALDI-TOF (/TOF)-MS (Bruker Autoflex Speed) with Nd:YAG lasers (355 nm) and smart beam system. All MS experiments were performed in the reflector positive ion mode, with 2000 laser shots per analysis and 5 independent experiments per sample. Data processing Comments:MS data preprocessing was carried out on python version 3.8, including peak detection, peak filtration, standardization, and batch effect removal by ComBat algorithm. The heatmap, hierarchical clustering analysis, Pearson correlation analysis and PCA analysis were performed on MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/). Machine learning and t-SNE clustering were conducted on Orange (version 3.25.0, the Bioinformatics Lab at University of Ljubljana, Slovenia). Software/procedures used for feature assignments: Python |
| Ion Mode: | POSITIVE |
