After euthanization, we collected bone marrow cells from 6-week-old male wild-type mice and cultured them in alpha minimum essential medium (α-MEM, Mediatech, Inc.) containing 100 U ml−1 penicillin (Sigma-Aldrich), 100 μg ml−1 streptomycin sulfate (Sigma-Aldrich) and 20% lot-selected fetal bovine serum (FBS, Atlanta Biologicals) at 37 °C in a 5% CO2 humidified incubator. After 72 h, we removed non-adherent cells and cultured adherent cells for an additional 7 d with a single media change. Then, we incubated cell aliquots for 20 min at 4 °C with fluorescein isothiocyanate-, phycoerythrin-, peridinin chlorophyll protein- and allophycocyanin-conjugated antibodies to mouse CD29 (Biolegend, HMβ1-1), Sca-1 (Biolegend, D7), CD45 (Biolegend, 30-F11) and CD11b (Biolegend, M1/70). We performed acquisition on a FACS Aria model (BD Biosciences), and did the analysis using FACS DIVE software version 6.1.3 (BD Biosciences). We sorted CD29+Sca-1+CD45−CD11b− cells and enriched them by further culture.
Hmβ1 1
Manufactured by BioLegend
Sourced in United States
HMβ1-1 is a monoclonal antibody that binds to the Human CD27 (TNFRSF7) protein. CD27 is a member of the tumor necrosis factor receptor superfamily and is expressed on T cells, B cells, and natural killer cells.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin sulfate, by Merck Group (2 mentions)
Facs aria model, by BD (2 mentions)
Facs dive software version 6, by BD (2 mentions)
M1 70, by BioLegend (2 mentions)
Penicillin, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions)
Nystatin, by Merck Group (1 mentions)
Streptavidin maleimide, by Merck Group (1 mentions)
Libs 2, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Cd11b m1 70, by BD (1 mentions)
Cd45 30 f11, by BD (1 mentions)
Cd31 mec 13, by BD (1 mentions)
Cd44 im7, by Thermo Fisher Scientific (1 mentions)
C kit 2b8, by BioLegend (1 mentions)
Cd34 mec14, by BioLegend (1 mentions)
Sca1 d7, by BioLegend (1 mentions)
Cd86 2331 fun 1, by BD (1 mentions)
4 protocols using hmβ1 1
1
Isolation and Characterization of Murine Mesenchymal Stem Cells
2
Biotinylated Fibronectin Binding Assay
3
Isolation and Characterization of Murine Mesenchymal Stem Cells
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After euthanization, we collected bone marrow cells from 6-week-old male wild-type mice and cultured them in alpha minimum essential medium (α-MEM, Mediatech, Inc.) containing 100 U ml−1 penicillin (Sigma-Aldrich), 100 μg ml−1 streptomycin sulfate (Sigma-Aldrich) and 20% lot-selected fetal bovine serum (FBS, Atlanta Biologicals) at 37 °C in a 5% CO2 humidified incubator. After 72 h, we removed non-adherent cells and cultured adherent cells for an additional 7 d with a single media change. Then, we incubated cell aliquots for 20 min at 4 °C with fluorescein isothiocyanate-, phycoerythrin-, peridinin chlorophyll protein- and allophycocyanin-conjugated antibodies to mouse CD29 (Biolegend, HMβ1-1), Sca-1 (Biolegend, D7), CD45 (Biolegend, 30-F11) and CD11b (Biolegend, M1/70). We performed acquisition on a FACS Aria model (BD Biosciences), and did the analysis using FACS DIVE software version 6.1.3 (BD Biosciences). We sorted CD29+Sca-1+CD45−CD11b− cells and enriched them by further culture.
4
Isolation and Characterization of Mouse Bone Marrow Mesenchymal Stem Cells
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Six- to eight-week-old DBA1J mouse bone marrow cells were collected by flushing femurs and tibias with Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA, USA) containing 2 mM L-glutamine (Gibco), 1% antibiotics (penicillin (10 U/ml)–streptomycin (10 g/ml)) (Gibco) and 15% heat-inactivated fetal bovine serum (FBS, Gibco) with an endotoxin level ≤5 EU/ml and hemoglobin level ≤10 mg/dl (Gibco)52 (link). When cells reached around 80% confluency, the medium was aspirated and 3–5 ml trypsin-EDTA (Gibco) were added to each dish. The dishes were then incubated for ~5 min to allow cell detachment. Next, an equal volume of culture medium was added to inactivate trypsin. The marrow cell immunophenotypes were persistently positive for Sca-1 (D7; BioLegend, San Diego, CA, USA), CD44 (IM7; eBioscience, San Diego, Ca, USA), and CD29 (HMβ1-1; BioLegend), but negative for c-Kit (2B8; BioLegend), CD11b (M1/70; BD Pharmingen, San Diego, CA, USA), CD34 (MEC14.7; BioLegend), CD106 (429 (MVCAM.A); BD Pharmingen), CD45 (30-F11; BD Pharmingen), CD31 (MEC 13.3; BD Pharmingen), CD80 (16-10A1, BD Pharmingen), and CD86 (2331 (FUN-1), BD Pharmingen) after more than 10 passages (two months of culturing) (Supplementary Fig. 1 ).
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