Computational protocol: Human Adipose-Derived Mesenchymal Stem Cells Cryopreservation and Thawing Decrease α4-Integrin Expression

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Protocol publication

[…] All samples were analyzed for the expression of surface markers (), using monoclonal antibodies against cluster of differentiation (CD) antigens, conjugated with fluorochromes and analyzed, before the cryopreservation and after thawing, by flow cytometer. Cells (number) were incubated with the antibodies that are described in and corresponding isotypes were used. The primary antibodies were incubated for 10 minutes in the dark. The antibody against CD105 is a purified antibody and it is not conjugated with fluorescence; for this reason, it was necessary to incubate it with a secondary antibody for more 15 minutes in the dark room and redo the cell washing step. Further 5 μL of 7-AAD was added and incubated in the dark room for 15 minutes. After that, 400 μL of binding buffer was added and the samples were analyzed by flow cytometer (FACS Calibur; Becton Dickinson, San Diego, USA) [, , ]. Prior to each test, the equipment was calibrated using BD Calibrite (Becton Dickinson, San Diego, USA), according to the manufacturer's instructions. All antibodies were used according to the manufacturer's instructions. 20.000 events (cells) were captured; the percentage values of each marker were analyzed through specific analysis software: Cyflogic version 1.2.1. [...] The obtained results in this study were expressed as mean ± standard deviation (SD). For the comparison between the molecules expression analysis before and after cryopreservation, the t-test was performed; p < 0.05 was considered significant. The data were organized in Excel spreadsheet and were analyzed with Statistica version 9.0 software. […]

Pipeline specifications

Software tools Cyflogic, Statistica
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens