I tried to get the epithelial cells from the tracheal mucosa. I incubated the tissues in S-MEM with 0.1% protease ⅩⅣ and 0.01%DNase at 4 ℃ overnight. But on the next day, I can only collect little cells. Does anyone else have experience to share?
The detailed method depends on which subclone you will focus on; nasal epithelial stem cells (NESCs), tracheal airway stem cells (TASCs), and distal airway stem cells (DASCs). But the important markers are anyway p63 and keratin5, 14. That is why flow cytometry with the antibody for stem cell surface markers enables you to isolate the stem cells among the scattered airways and alveolar cells.
Second method is that airway cells were cultivated onto a feeder layer of lethally irradiated 3T3-J2 cells in cFAD media and clonal analysis was based on previously described methods for epidermal stem cells (Barrandon and Green, 1987). Importantly, usage of 3T3-J2 as feeder cells selects stem cells without flow cytometry,
There is a good protocol for doing this in human lung here: https://www.med.unc.edu/cfpulmcenter/files/Book%20Chapter.pdf
I would suggest that as a good starting point.
Best wishes for a successful outcome!
Thank you for your answers, I will consider all the suggestions and try again.
Best wishes
Use the Georgetown protocol
ROCK Inhibitor and Feeder Cells Induce the
Conditional Reprogramming of Epithelial Cells, Liu et al
Am J Pathol 2012, 180:599–607; DOI: 10.1016/j.ajpath.2011.10.036
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