To obtain fetal liver cells, one male mouse was placed with two female mice during the light period and left overnight. At the indicated dates, pregnant females were sacrificed by CO2 asphyxia, and fetal liver cells from individual embryos were surgically isolated, minced, and passed through 70 μm cell strainer (Cat. 352350; BD, New Jersey, USA). For adult mice, total BM was harvested by crushing long bones and spine, while spleens were dissected and single-cell suspensions obtained by pressing through a 70 μm cell strainer. Red blood cells were lysed with ammonium-chloride potassium (ACK) lysis buffer (150 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA, pH 8.0) for 10 min on ice. Lineage depletion was achieved according to the manufacturers’ protocol of mouse hematopoietic lineage depletion kit (Cat. 130-090-858; Miltenyi Biotech, Bergisch Gladbach, Germany). Cytospin preparations of approximately 105 cells were made by centrifugation for 3 min at 300 r.p.m. using a Shandon Cytospin 3 centrifuge using cytofunnel disposable sample chambers (Cat. 5991040; Thermo Fisher Scientific, Reinach, Switzerland) and non-coated cytoslides (Cat. 5991051; Thermo Fisher Scientific, Reinach, Switzerland). Cytospots were stained with Wright Giemsa solution.
Mouse hematopoietic lineage depletion kit
Manufactured by Miltenyi Biotec
Sourced in Germany
The Mouse Hematopoietic Lineage Depletion Kit is a laboratory tool used to selectively remove specific cell types from mouse hematopoietic cell populations. It allows for the isolation and enrichment of target cell populations by depleting unwanted lineages.
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2 protocols using mouse hematopoietic lineage depletion kit
1
Isolation and Staining of Fetal and Adult Murine Hematopoietic Cells
2
Flow Cytometry Analysis of Hematopoietic Cells
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Flow cytometry (FACS analysis) and cell sorting Single cell suspensions prepared from BM, spleen, thymus, lymph node, peripheral blood, and E14.5 fetal livers were tested after removal of red blood cells by ACK lysis buffer. Cells were blocked with purified anti-mouse CD16/32 and then stained with appropriate fluorochrome-conjugated antibodies (See key resources table above) for 30 min on ice in staining buffer (phosphate buffered saline with 2% fetal bovine serum). Details of antibody combinations used to identify each population are given in the figure legends.
After staining, the cells were washed with staining buffer. BD FACS Calibur and Beckman CytoFLEX were used for analysis. Data were analyzed with Flowjo software.
For the intracellular staining (Ki67 and gH2AX staining), BM cells were stained with HSC cell surface maker first, then fixed and permeabilized using the Fixation/Permeabilization Solution Kit, followed by Brilliant Violet 605-Ki67 (cell cycles) and Alexa Fluor 488-H2A.X (DNA damage) staining, and further incubated with DAPI.
For cell sorting, BM cells were depleted using the mouse hematopoietic lineage depletion kit (Miltenyi Biotec) to enrich Lin -cells. The enriched Lin -cells were then stained with anti-lineage cocktail, anti-Scal-1, and anti-cKit antibodies for further sorting to enrich HSPCs (LSK cells). BD FACS Aria was used for cell sorting.
After staining, the cells were washed with staining buffer. BD FACS Calibur and Beckman CytoFLEX were used for analysis. Data were analyzed with Flowjo software.
For the intracellular staining (Ki67 and gH2AX staining), BM cells were stained with HSC cell surface maker first, then fixed and permeabilized using the Fixation/Permeabilization Solution Kit, followed by Brilliant Violet 605-Ki67 (cell cycles) and Alexa Fluor 488-H2A.X (DNA damage) staining, and further incubated with DAPI.
For cell sorting, BM cells were depleted using the mouse hematopoietic lineage depletion kit (Miltenyi Biotec) to enrich Lin -cells. The enriched Lin -cells were then stained with anti-lineage cocktail, anti-Scal-1, and anti-cKit antibodies for further sorting to enrich HSPCs (LSK cells). BD FACS Aria was used for cell sorting.
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