Subcutaneous inguinal adipose tissue was removed from SD rats, finely minced, and enzymatically digested at 37 °C in minimum essential media (MEM) containing 0.1% collagenase type I (Sigma-Aldrich, St Quentin Fallavier, France) for 30 min (min), 3 times. The digested tissue was filtered through a 100 µm filter. Collagenase was then neutralized with culture medium containing 10% fetal bovine serum (FBS). After centrifugation (1200 rpm for 3 min), cells were suspended in MEMα containing 20% FBS, penicillin–streptomycin and l -glutamin (all from Invitrogen) plated at 1000 cells by cm2 and cultured at 37 °C in humidified 5% CO2. After 6 d, the monolayer of adherent cells was trypsinized, washed in PBS 1× 3 times before injection in rats. The phenotype of amplified Ad-MSCs was verified by flow cytometry. The percentage of CD90 (clone OX-7; BD Biosciences) and CD73 (clone 5F/B9; BD Biosciences) positive cells were analyzed, and the absence of hematopoietic cells was verified with CD34 (clone ICO115, Santa Cruz Biotechnology) and CD45 (clone OX-1; BD Biosciences) markers. Isotype identical antibodies served as controls.
Cd90 clone ox 7
Manufactured by BD
Sourced in France
CD90 (clone OX-7) is a cell surface antigen commonly used as a marker for specific cell types. It functions as a glycophosphatidylinositol (GPI)-anchored protein on the cell membrane. CD90 is expressed on various cell types, including mesenchymal stem cells, neurons, and hematopoietic stem cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd73 clone 5f b9, by BD (3 mentions)
Cd34 clone ico115, by Santa Cruz Biotechnology (3 mentions)
Cd45 clone ox 1, by BD (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
L glutamin, by Thermo Fisher Scientific (1 mentions)
Facsort, by BD (1 mentions)
3 protocols using cd90 clone ox 7
1
Isolation and Characterization of Rat Adipose-Derived Mesenchymal Stem Cells
2
Adipose-derived MSC Isolation and Characterization
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Adipose-derived MSCs were obtained by digesting the subcutaneous inguinal adipose tissue of seven-week-old GFP-transgenic SD rats as previously described10 . The phenotype of amplified Ad-MSCs was verified by flow cytometry. The percentage of CD90 (clone OX-7; BD Biosciences, Le pont de Claix, France) and CD73 (clone 5F/B9; BD Biosciences) positive cells were analyzed and the absence of hematopoietic cells was verified with CD34 (clone ICO115, Santa Cruz, Dallas, Texas, USA) and CD45 (clone OX-1; BD Biosciences) markers. Isotype identical antibodies served as controls. The potential of adipogenic and osteogenic differentiations was also verified before injection. Cells were plated at 10 000 cells per cm2, at 80% of confluence, inductive media were applied during 3 weeks. For osteogenic differentiation: MEMα (Minimum Essential Medium) 10% FCS (foetal calf serum), 10 mM β–glycerophosphate, 10−7 M Dexamethasone, 50 µg/ml L-ascorbic Acid. For adipogenic differentiation: MEMα 10% SVF, 100 µM Isobutyl methylxanthine, 10−7 M Dexamethanone, 60 µM Indomethacin, 10 µg/ml Insulin.
3
Isolation and Characterization of Rat Bone Marrow-Derived MSCs
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Bone marrow-derived MSCs were obtained by flushing femurs of 7-wk-old rats, humanely euthanized as described previously [4] . In brief, after 10 d, the monolayer of adherent cells (P0) was seeded at 5000 cells/cm 2 (P1). At each passage, the expression of MSC markers was studied by FACS, with FACSort (BD Biosciences, Le Pont-de-Claix, France). Flow cytometry analysis confirmed that the cells were MSCs by identifying the following cell surface markers: CD90 (clone OX-7; BD Biosciences) and CD73 (clone 5F/B9; BD Biosciences). The absence of hematopoietic cells was verified with CD34 (clone ICO115; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and CD45 (clone OX-1; Becton Dickinson, Le Pont-de-Claix, France, France) markers. On average, 95% of the MSCs expressed CD90, 81% CD73, 2% CD34, and 6.4% CD45. Identical isotope antibodies served as controls. The potential for adipogenic, osteogenic, and chondrogenic differentiation and the ability to form CFU fibroblasts were also analyzed. FPC-MSCs were obtained by incubating the MSCs with flagellin (1 mg/ml) for 24 h before use.
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