I have to isolate T cells from C57Bl/6 mouse skin, in particular the epidermis, where tissue resident T memory cells are found. Since I am more used to isolating cells from deep tissues like liver and spleen, the mouse skin has shown to be a lot more challenging to isolate viable T cells from. I was wondering if anyone experienced in skin digestion and single cell suspension generation could recommend suitable published protocols. I want to run FACS analysis on these preps to characterize T cells present in the skin during infection. Thanks for the help.
I will write up the brief protocol to isolate the epidermis and prepare for FACS.
Day1
1) to shave the dorsal skin hair and cut the skin off
2) to make the skin sterile with povidone iodine for 5 minutes and 70% ethanol for 10 minutes for twice, and then to wash the skin section with PBS for three times (15 minutes)
3) to incubate the skin in the total mixture of 0.2% trpsin and 0.02％EDTA (1:1) under 4 degree overnight
Day2
4) The next day, to put in 37 degree for 30 minutes
5) to exfoliate the epidermis, which looks like the remnant black hair
6) to filter the epidermis in 20% serum-containing PBS using 50 ml falcon tube
7) to centrifuge X1000 for 5 minutes
8) to suspend with 20% serum-containing PBS, the concentration of which is sufficient for FACS analysis
I have looked into isolating T-cells from dorsal shaved skin of Balb/c mice. Fatty tissue is your main enemy. But regardless of the protocol used, my major concern with all of these protocols is this: How representative are the T-cells that are isolated? Are these enriched for epidermal T-cells versus dermal T-cells?
I found that the best results (used by most good publications on skin T-cells) generally isolate T-cells from the murine ears (no shaving, no fat, easy to handle).
Good luck.
I would suggest you to look into Clark RA publications, she works with human skin T cells and even developed a method to amplify memory skin derived T cells in vitro, so I am pretty sure you may find the right protocol among her papers.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques