Isolation and culture of mouse keratinocytes
Isolation and culture of mouse keratinocytes
This protocol is a modification of the force method of Hennings et al. (1980). Gestational BALB/c mice are available from Harland Sprague Dawley, and the protocol is also applicable to the isolation of primary rat keratinocytes.
Operation method
Isolation and culture of mouse keratinocytes Move 1) Asphyxiate 1~2 days old neonatal nude mice and place them in a petri dish. Wash the nude mice with 1% iodine polyvinylpyrrolidone solution, rinse them thoroughly under running water, and then wash them twice with 70% ethanol. For more product details, please visit Aladdin Scientific website.
2) Remove the limbs and tails of the mice.
3) Make a simple incision in the skin of the mouse from the tail to the nose and gently peel the skin from the entire body using rat tooth forceps. skin.
4) Place the exfoliated skin tissue on the surface of a sterile petri dish placed on ice until all the skin has been collected.
5) Hold the skin tissue with 2 rat dental forceps, rinse the skin tissue (dermal tissue underneath) in a sterile petri dish of HBSS containing 2.5% trypsin, and then place it at 4°C overnight. The epidermis should not be submerged in the trypsin solution.
HBSS
NaCl 8 g/L
KCl 400 mg/L
KH 2 PO4 60 mg/L
Anhydrous Na 2 HPO4 47.86 mg/L
Anhydrous Glucose 1000 mg/L
NaHCO3 350 mg/L
6) Remove the tissues from the trypsin-treated solution, and place the epidermis face-down on the surface of a dry, sterile cell culture dish.
7 ) Carefully cut the epidermal residue with scissors and place it in a sterile flask containing "regular" culture medium (MEM with 10% FCS, 100IU/ml penicillin, 100ug/ml streptomycin, 2~4 mmol/L L-glutamine; 10 ml for each 5 mouse skins) with a magnetic stirrer for 45 min at 37°C.
8) Place the tissue in a dry sterile cell culture dish with 4 layers of sterile Nitex gauze (16xx wells, provided by Martin). 16xx wells, provided by Martin) to filter the liquid.
The stratum corneum cells remain on the surface of the gauze while other cells pass through.
9) Wash the cells down with culture medium, the amount of medium should be about 10 ml for one piece of skin, and inoculate the cells into plastic cell culture flasks or glass coverslips and incubate them for 4-12 hours at 37°C. The cells can also be centrifuged and the sediment can be collected.
Alternatively, the cells can be centrifuged to collect the precipitate (800 g, 10 min), resuspended in culture medium containing 10% DMSO, and frozen in liquid nitrogen.
10) Observe the cell populations on the surface of the culture dish after 4-12 hours.
11) Transfer the cells to a low-calcium culture medium, and the keratinocytes will begin to multiply.