After harvesting stem cells from adipose tissue, we
evaluated their mesenchymal characteristics using two
methods: flow cytometry and differentiation into the
osteoblastic lineage.
Phenotyping of cell surface markers for MSCs at
the second passage involved labeling with anti-human
antibodies, including CD90, CD44, CD10, CD106,
CD34, and CD45, using fluorescein isothiocyanate
(FITC) (Beckman Coulter, Fullerton, CA). Human
isotype antibodies served as controls (Becton Dickinson;
Beckman Coulter).
For differentiation characterization, a density of 5×104cells/ml at the second passage was cultured in a sixwell
plate with DMEM and 10% FBS. Upon reaching
confluence within one week, the medium was replaced with
osteogenic differentiation media to assess the cells’ ability
to differentiate into the osteoblastic lineage. This medium
comprised DMEM, 50 μg/ml ascorbic acid 2-phosphate, 10
nM dexamethasone, and 10 mM ƒÀ-glycerol phosphate (all
obtained from Gibco, United States). Incubation occurred
at 37°C with 5% CO2 for 21 days, with medium renewal
every three days. Subsequently, the cells were fixed with
10% formalin for 10 minutes, followed by staining with
Alizarin Red for 20 minutes at room temperature. Finally,
the cells were examined under an optical microscope, and
photographic documentation was undertaken (22 (link)).
evaluated their mesenchymal characteristics using two
methods: flow cytometry and differentiation into the
osteoblastic lineage.
Phenotyping of cell surface markers for MSCs at
the second passage involved labeling with anti-human
antibodies, including CD90, CD44, CD10, CD106,
CD34, and CD45, using fluorescein isothiocyanate
(FITC) (Beckman Coulter, Fullerton, CA). Human
isotype antibodies served as controls (Becton Dickinson;
Beckman Coulter).
For differentiation characterization, a density of 5×104cells/ml at the second passage was cultured in a sixwell
plate with DMEM and 10% FBS. Upon reaching
confluence within one week, the medium was replaced with
osteogenic differentiation media to assess the cells’ ability
to differentiate into the osteoblastic lineage. This medium
comprised DMEM, 50 μg/ml ascorbic acid 2-phosphate, 10
nM dexamethasone, and 10 mM ƒÀ-glycerol phosphate (all
obtained from Gibco, United States). Incubation occurred
at 37°C with 5% CO2 for 21 days, with medium renewal
every three days. Subsequently, the cells were fixed with
10% formalin for 10 minutes, followed by staining with
Alizarin Red for 20 minutes at room temperature. Finally,
the cells were examined under an optical microscope, and
photographic documentation was undertaken (22 (link)).
