We characterized the primary cultured cells from human adipose tissues. The cells were observed their morphology by inverted microscope and characterized using surface markers and fluorescence-activated cell sorting (FACS) analyses, with labeling by fluorescein-isothiocyanate (FITC)-conjugated monoclonal antibodies directed against the cluster of differentiation (CD) markers (BD Biosciences Pharmingen, San Diego, CA) CD73, CD90 or CD105 or with phycoerythrin (PE)-conjugated monoclonal antibodies directed against CD31, CD34 or CD45. Analysis was performed using a FACscan argon laser cytometer (Becton Dickinson, San Jose, CA). Additionally, the multipotency of the cells was determined using a mesenchymal differentiation kit (Invitrogen) as previously described [10 (link)]. The differentiation of hAT-MSCs into adipocytes, osteocytes and chondrocytes was confirmed by Oil Red O, Alizarin Red S and Alcian blue staining, respectively. All experiments were conducted in triplicate.
Fitc conjugated monoclonal antibodies
Manufactured by BD
FITC)-conjugated monoclonal antibodies are laboratory reagents used in various immunoassays and flow cytometry applications. The antibodies are labeled with the fluorescent dye fluorescein isothiocyanate (FITC), which allows for the detection and identification of specific target molecules or cells.
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2 protocols using fitc conjugated monoclonal antibodies
1
Characterization of Human Adipose-Derived Cells
2
Phenotypic Characterization of ADSCs
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Flow cytometry was done to confirm that the used undifferentiated ADSCs maintained their phenotypic characterization after the third passage of growth in culture. Expression of the cell surface markers CD 44, CD45, and CD73 was quantified. Briefly, trypsin-harvested ADSCs were washed with PBS three times, and aliquots of 105 cells were incubated with phycoerythrin-conjugated monoclonal antibodies directed against CD45 (eBioscience Cat # 12-0459), CD73 (BD Pharmingen Cat # 550257) or FITC conjugated monoclonal antibodies directed against CD44 (BD Cat # 348057). Chemicals were purchased from Sigma-Aldrich (2nd St #3306, St. Louis, MO 63118, United States).
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