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Published on: April 29, 2016
Aldefluor protocol to sort keratinocytes stem cells from skin
Samuel Marcos Ribeiro Noronha1, Alfredo Gragnani2, Thiago Antônio Calado Pereira3
1PhD, Postdoctoral Researcher, Division of Plastic Surgery, Universidade Federal de São Paulo (UNIFESP), Brazil. Conception and design of the study, manuscript preparation , final approval.
Purpose:
To investigate the use Aldefluor® and N, N - Dimethylaminobenzaldehyde (DEAB) to design a protocol to sort keratinocyte stem cells from cultured keratinocytes from burned patients.
Methods:
Activated Aldefluor® aliquots were prepared and maintained at temperature between 2 to 8°C, or stored at -20°C. Next, the cells were collected following the standard protocol of sample preparation.
Results:
Best results were obtained with Aldefluor® 1.5µl and DEAB 15 µl for 1 x 106 cells, incubated at 37°C for 15 minutes. Flow cytometer range for keratinocyte stem cells separation was evaluated. There were 14.8% of stem cells separated in one sample of keratinocyte culture used to pattern the protocol. After being defined the ideal concentration, the same test pattern was performed in other keratinocyte samples. We observed a final mean of 10.8%.
Conclusion:
Aldefluor® has been shown as a favorable marking of epidermal keratinocyte stem cells for subsequent separation on a flow cytometer, with detection of 10.8% of epidermal keratinocyte stem cells, in this protocol.
