Murine bone marrow MSCs were identified and isolated using flow cytometry analyses and FACS sorting as described previously.36 (link),64 (link) At the time of euthanasia, bone marrow cells were flushed from femoral and tibial medullary cavities. Cell numbers were determined after removal of red blood cells with ammonium chloride–potassium lysis buffer (Quality Biological). Non-permeabilized cells were incubated with the antibodies and fixed in 1% paraformaldehyde. The antibodies used for flow cytometry identification of MSCs included anti-CD45-APC (Biolegend, 103112, 1:200), anti-Ter119-APC (Biolegend, 116212, 1:200), anti-CD31-APC (Biolegend, 102510, 1:100), and anti-LepR-biotin (R&D Systems, AF497, 1:500). Cells were stained with antibodies in staining buffer (HBSS + 2% fetal bovine serum) on ice for 1 h, and then washed with staining buffer. Biotin-conjugated antibody was incubated with streptavidin-brilliant violet 421 (Biolegend, 405225, 1:500) for another 20 min. LepR+CD45–CD31–Ter119- cells was analyzed using a BD LSR II flow cytometer or sorted using a BD FACSAria IIu cell sorter.
Anti lepr biotin
Manufactured by R&D Systems
Anti-LepR-biotin is a biotinylated antibody that recognizes the Leptin Receptor (LepR) protein. This product can be used for the detection and/or purification of LepR in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin brilliant violet 421, by BioLegend (2 mentions)
Anti cd45 apc, by BioLegend (1 mentions)
Anti cd31 apc, by BioLegend (1 mentions)
Facsaria iiu cell sorter, by BD (1 mentions)
Anti ter119 apc, by BioLegend (1 mentions)
Ammonium chloride potassium lysis buffer, by Quality Biological (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Anti cd45, by Thermo Fisher Scientific (1 mentions)
Anti ter119, by Thermo Fisher Scientific (1 mentions)
Anti sca 1, by Thermo Fisher Scientific (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Anti cd31, by Thermo Fisher Scientific (1 mentions)
Collagenase 1 and 4, by Worthington (1 mentions)
Facscelesta flow cytometer, by BD (1 mentions)
Apc brdu flow kit, by BD (1 mentions)
Dnase 1, by Worthington (1 mentions)
2 protocols using anti lepr biotin
1
Isolation of Murine Bone Marrow MSCs
2
Analyzing Bone Marrow Cell Populations
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Whole bone marrow cells were flushed using Hank’s balanced salt solution (HBSS) (Mg2+ and Ca2+ plus) and digested with 200 U/mL DNase I and 250 μg/mL Collagenase I and IV (Worthington) at 37°C for 30 min. Cell suspension in 100 μL HBSS with 2% bovine serum was stained with antibodies, including anti-LEPR-biotin (1:100, R&D Systems, BAF497), anti-CD45 (1:200, eBioscience, 30F-11), anti-CD31 (1:200, eBioscience, 390), anti-TER119 (1:200, eBioscience, TER-119), anti-SCA-1 (1:200, eBioscience, E13–161.7), and/or anti-PDGFRα-biotin (1:100, BioLegend, APA5), and then brilliant violet 421 streptavidin (1:500, BioLegend). Samples were analyzed using a FACSCelesta flow cytometer (BD Biosciences). Data were analyzed by FlowJo software.
For BrdU incorporation assays, mice were given an intraperitoneal injection of 1 mg BrdU (Sigma) per 6 g of body mass in PBS and maintained on 1 mg/mL of BrdU in the drinking water for 14 days. The frequency of BrdU+ cells was then analyzed by flow cytometry using the APC BrdU Flow Kit (BD Biosciences).
For apoptotic cell staining, cells were stained by anti-Annexin V antibody before flow cytometry following the manufacture's introductions (Thermo Fisher).
For BrdU incorporation assays, mice were given an intraperitoneal injection of 1 mg BrdU (Sigma) per 6 g of body mass in PBS and maintained on 1 mg/mL of BrdU in the drinking water for 14 days. The frequency of BrdU+ cells was then analyzed by flow cytometry using the APC BrdU Flow Kit (BD Biosciences).
For apoptotic cell staining, cells were stained by anti-Annexin V antibody before flow cytometry following the manufacture's introductions (Thermo Fisher).
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