Sufficient cryopreserved ADMSC vials were thawed to provide the required dose for administration. The frozen ADMSCs were thawed and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% human serum, 1% penicillin-streptomycin solution, and 1% stable glutamine (Biological Industries, Kibbutz Beit Haemek, Israel) at 37°C and 5% CO2 incubator as described before [16 ]. Briefly, ADMSCs were recovered and resuspended in physiological saline solution and transferred to the patient with the temperature-controlled bag. The morphology of cell appearance and viability were assessed besides the identification of phenotypic characteristics of the cells and microbiological tests. Immunophenotyping characterization of ADMSC was performed by using flow cytometry analyses (Beckman Coulter, USA) with antibodies against CD45, CD73, CD90, CD105, CD11b, CD19, CD34, and CD44 (BD Stem Flow hMSC kit).
Flow cytometry analyses
Manufactured by Beckman Coulter
Sourced in United States
Flow cytometry is an analytical technique used to measure and analyze the physical and chemical characteristics of cells or particles in a fluid sample. It involves passing the sample through a laser beam and detecting the scattered light and fluorescence signals from individual cells or particles. This data provides information about the size, granularity, and presence of specific molecules or markers on the surface or inside the cells.
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2 protocols using flow cytometry analyses
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Cryopreserved ADMSC Characterization and Administration
2
Transfection and Uptake Analysis Protocol
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In vitro transfection was performed as previously described.12 (link) Briefly, cells were trypsinized and seeded at 5×104 cells/well in three 24-well plates. The plates were left overnight to achieve 60%–80% confluence, and were then transfected with the predetermined formulations. After 6 hours, cells were analyzed for FAM-siRNA uptake using an inverted fluorescence microscope (IX71, Olympus Corporation, Tokyo, Japan). Cells were also trypsinized, centrifuged (2,000× g for 3 minutes), and resuspended in phosphate-buffered saline for flow cytometry analyses (Beckman Coulter, Fullerton, CA, USA).
For confocal laser scanning microscopy, MCF-7 cells were seeded at 5×104 cells/well in 35 mm glass bottom culture dishes (MatTek Corp, Ashland, MA, USA) and then incubated for 24 hours at 37°C in 5% CO2. Next, the culture medium was replaced with ACC/CaIP6/FAM-siAKT1 complexes in 500 µL of serum-free Dulbecco’s Modified Eagle’s Medium. Cells were then washed three times with phosphate-buffered saline at predetermined time intervals, and the nuclei were stained with 4′,6′-diamidino-2-phenylinodole (DAPI, Sigma-Aldrich, St Louis, MO, USA) for 5 minutes. The cells were directly observed using an Olympus FluoView confocal microscope and analyzed by FV10-ASW viewer software.
For confocal laser scanning microscopy, MCF-7 cells were seeded at 5×104 cells/well in 35 mm glass bottom culture dishes (MatTek Corp, Ashland, MA, USA) and then incubated for 24 hours at 37°C in 5% CO2. Next, the culture medium was replaced with ACC/CaIP6/FAM-siAKT1 complexes in 500 µL of serum-free Dulbecco’s Modified Eagle’s Medium. Cells were then washed three times with phosphate-buffered saline at predetermined time intervals, and the nuclei were stained with 4′,6′-diamidino-2-phenylinodole (DAPI, Sigma-Aldrich, St Louis, MO, USA) for 5 minutes. The cells were directly observed using an Olympus FluoView confocal microscope and analyzed by FV10-ASW viewer software.
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