Third-generation ADSCs were treated separately using the following antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-CD90, FITC-conjugated anti-CD45, FITC-conjugated anti-CD31, phycoerythrin (PE)-conjugated anti-CD34, and PE/Cy7-conjugated anti-CD29 PE-conjugated anti-CD-11b/c (Cyagen Biosciences, USA). Aliquots of cells were incubated with 2 µg/ml of antibody at 4°C in the dark for 30 min, washed twice with 350 µl of fluorescence-activated cell sorting (FACS) buffer, and centrifugation at 1000 ×g for 5 min. Cell pellets were gently resuspended in 100 µl of buffer and transferred to FACS tubes for analysis using single-channel flow cytometry (BD, USA); after which the percentage of positive cells were calculated for each antibody marker.
Single channel flow cytometry
Manufactured by BD
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Single-channel flow cytometry is an analytical technique used to measure and analyze the physical and chemical characteristics of cells or particles suspended in a fluid as they pass through a laser beam. It provides quantitative information about the size, granularity, and fluorescence intensity of individual cells or particles within a sample.
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3 protocols using single channel flow cytometry
1
Characterization of Third-Generation ADSCs
2
Comprehensive ADSC Phenotypic and Differentiation Assay
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For analyzing the stem cell phenotype, third-passage ADSCs were labeled with fluorescein isothiocyanate- (FITC-) conjugated antibodies against CD90, CD45, CD44, CD34, CD29, and CD11 isoforms b, 2f, and c (Cyagen Biosciences). Other ADSC cultures were stained with FITC-conjugated mouse IgG or FITC-conjugated hamster IgG (Cyagen Biosciences) as a negative control. Expression distribution was assessed by single-channel flow cytometry (BD Biosciences, USA).
Differentiation potential was assessed as previously described [1 (link)]. Briefly, third-generation ADSCs were used for testing adipocyte differentiation potential in an adipogenic differentiation medium (Cyagen Biosciences). After 9 days in culture, cells were stained with the lipid marker oil red O. Osteogenic differentiation was tested by staining with the calcium deposition dye alizarin red after 3 weeks in an osteogenic differentiation medium (Cyagen Biosciences). For chondrocyte differentiation, mesenchymal stem cells were incubated for 26 days in a complete chondrocyte differentiation medium (Cyagen Biosciences), following which they were fixed with formalin, embedded in paraffin, mounted on slides, and stained with the chondrocyte matrix marker alcian blue.
Differentiation potential was assessed as previously described [1 (link)]. Briefly, third-generation ADSCs were used for testing adipocyte differentiation potential in an adipogenic differentiation medium (Cyagen Biosciences). After 9 days in culture, cells were stained with the lipid marker oil red O. Osteogenic differentiation was tested by staining with the calcium deposition dye alizarin red after 3 weeks in an osteogenic differentiation medium (Cyagen Biosciences). For chondrocyte differentiation, mesenchymal stem cells were incubated for 26 days in a complete chondrocyte differentiation medium (Cyagen Biosciences), following which they were fixed with formalin, embedded in paraffin, mounted on slides, and stained with the chondrocyte matrix marker alcian blue.
3
Isolation and Characterization of Rat BMSCs
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