Human foreskin fibroblasts (HF; kindly provided by Dr. Gregory Pari) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA). Human BM cells were harvested from healthy adult donors after informed consent according to guidelines from the Office of Human Research Protection at the University of Nevada at Reno. Donors were determined in a commercial laboratory to be CMV-seronegative or -seropositive by testing for antibodies to CMV. Low-density BM mononuclear cells were separated using a Ficoll-Hypaque density gradient (1.077 g/mL; Sigma, St. Louis, MO) washed twice in Iscove’s modified Dulbecco’s media (Invitrogen) with penicillin (100 U/mL), streptomycin (100 mg/mL) and amphotericin B (0.25 mg/mL; Gibco Laboratories, Grand Island, NY). Stro-1+ cells were selected by magnetic cell sorting (Miltenyi Biotec, Inc., Auburn, CA) using a Stro-1 antibody (R&D Systems, Minneapolis, MN) as previously described (31 (link)). Human fetal LVR, BRN and LNG (18–22 weeks gestational age) were purchased from Advanced Bioscience Resources (Alameda, CA) and sorted using Stro-1 antibody (R&D Systems) as previously described (30 (link),31 (link)). The purity of the sorted cells exceeded 95% and in addition to CD29+, CD44+, CD73+, CD90+, CD146+, NG2+ cells maintained Stro-1, throughout culture.
Stro 1 antibody
Manufactured by R&D Systems
Sourced in United States
Stro-1 antibody is a mouse monoclonal antibody that recognizes a cell surface antigen expressed on human mesenchymal stem cells. It is commonly used as a marker for the identification and isolation of mesenchymal stem cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Magnetic bead cell sorting, by Miltenyi Biotec (2 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Ficoll hypaque density gradient, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Iscove s modified dulbecco s media, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Accuri c6, by BD (1 mentions)
Cflow plus software, by BD (1 mentions)
Streptavidin percp cy5, by BD (1 mentions)
Aria flow cytometer, by BD (1 mentions)
Streptavidin pacific blue, by Thermo Fisher Scientific (1 mentions)
Percp cy5, by BD (1 mentions)
Caltag, by Thermo Fisher Scientific (1 mentions)
Color conjugated mouse igg1k, by BD (1 mentions)
Ficoll density gradient, by Merck Group (1 mentions)
Iscove s modified dulbecco s medium, by Thermo Fisher Scientific (1 mentions)
5 protocols using stro 1 antibody
1
Isolation and Culture of Human Mesenchymal Stem Cells
2
Multimarker Characterization of Mesenchymal Stem Cells
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Cells were collected and washed with PBS. Cells were then incubated with PE‐coupled antibodies against CD34, CD44, CD90, CD105, CD166 (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA) and FITC‐coupled antibody against CD146 (Becton Dickinson Biosciences). For the examination of STRO‐1 marker, cells were incubated with STRO‐1 antibody (R&D Systems, Minneapolis, MN, USA) for 1 hour on ice. After washing, cells were incubated with PE‐conjugated secondary antibody (R&D Systems) for 30 minutes on ice, and then washed with PBS.19 , 20 Fluorescence was detected using a BD Accuri C6 (Becton Dickinson Biosciences). Data were analysed using CF Low Plus Software (Becton Dickinson Biosciences).
3
Isolation and Expansion of Stro-1+ Bone Marrow Mesenchymal Cells
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Human bone mononuclear cells (BMNCs) were obtained from AllCells (Alameda, CA). BMNCs were enriched for the Stro-1+ fraction using a Stro-1 antibody (R&D Systems, Minneapolis, MN) and magnetic bead cell sorting (Miltenyi Biotec, Inc., Auburn, CA). Stro-1+ cells were expanded in vitro at 37℃ in 5% CO2 humidified air, in MSC-GM (growing media). At confluence, cells were detached with 0.25% trypsin (Invitrogen Corp., Carlsbad, CA), and trypsin was neutralized with media containing 10% fetal bovine serum (Thermo Fisher Scientific, Grand Island, NY). Characterization by flow cytometry, and by functional studies, demonstrated that these cells displayed markers characteristic of bone marrow-derived mesenchymal cells/pericytes, including CD105, CD146, CXCL12, CD90, CD44, and CD29, and that they were able to undergo trilineage differentiation into adipocytes, cartilage, and bone.14 (link) Cells were cultured in T-75 flasks using 36 mL of media per flask, incubated at 37℃, with 5% CO2, and grown to 100% confluency before being exposed to the ELF-EMF.
4
Characterization of Mesenchymal Stem Cells by Flow Cytometry
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The characteristics of the BMSCs were evaluated using flow cytometry in a previous study [7 (link)]. Aria flow cytometer (Becton Dickinson, San Jose, CA, USA) and the following antibodies were used: fluorescence isothiocyanate (FITC)-conjugated, phycoerythrin-conjugated, peridinin-chlorophyll-protein-conjugated (PerCP-Cy5.5), allophycocyanin-conjugated, Alexa Fluor 405-conjugated, or biotinylated antibodies against HLA-ABC, HLA-DR, CD3, CD14, CD19, CD34, CD73, CD90, CD106, CD146, CC chemokine receptor-5 (all from BD Pharmingen, Franklin Lakes, NJ, USA), CD10, CD29 (both from Dako, Glostrup, Denmark), and CD45 (Caltag; Invitrogen). Unconjugated CD105 antibody (Immunotech, Marseille Cedex 9, France) was covalently conjugated to FITC. The STRO-1 antibody (R&D Systems, Minneapolis, MN, USA) was detected using phycoerythrin-conjugated anti-mouse IgM. Biotinylated antibodies were detected using streptavidin Pacific Blue (Molecular Probes, Invitrogen, Carlsbad, CA, USA) or streptavidin PerCPCy5.5 (BD Pharmingen) conjugates. Propidium iodide (Dojindo) was used to detect dead cells. Color-conjugated mouse-IgG1k (BD Pharmingen) was used as a negative control. Data analysis was performed using the FlowJo software (TreeStar, San Carlos, CA, USA).
5
Isolation and Expansion of Stro-1+ Mesenchymal Cells
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Heparinized human BM was obtained from healthy donors after informed consent according to guidelines from the Office of Human Research Protection at Wake Forest University Health Sciences. Low‐density BM mononuclear cells (BMNC) were separated by Ficoll density gradient (1.077 g/ml Sigma‐Aldrich, St. Louis, MO) centrifugation and washed twice in Iscove's modified Dulbecco's medium (Invitrogen, Carlsbad, CA). BMNC were first enriched for the Stro‐1+ fraction using a Stro‐1 antibody (R&D Systems, Minneapolis, MN) and magnetic bead cell sorting (Miltenyi Biotec, Inc., Auburn, CA). Stro‐1+ cells were expanded in vitro at 37°C in 5% CO2 humidified air, in MSCGM, using Fibronectin‐coated tissue culture flasks. At a confluence of 60%–75%, cells were detached with 0.25% trypsin (Invitrogen Corp., Carlsbad, CA) for 3–5 minutes at 37°C. trypsin was neutralized with media containing 10% fetal bovine serum (Thermo Fisher Scientific, Grand Island, NY), and cells were passaged at a 1:3 ratio into gelatin‐coated tissue culture flasks in MSCGM. Characterization by flow cytometry, and by functional studies, demonstrated that these cells displayed markers characteristic of BM‐derived mesenchymal cells/pericytes, including CD105, CD146, CXCL12, CD90, CD44, and CD29, and that they were able to undergo trilineage differentiation into adipocytes, cartilage, and bone 28 .
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