Experimental reagent

HBSS:
NaCl 8g/L
KCl 400mg/L
KH2PO4 60mg/L
Anhydrous Na2PO4 47.86mg/L
Anhydrous glucose 1000mg/L
NaHCO3 350mg/L

Experimental Materials

Pregnancy BALB/c mice

Experimental procedure

1. After suffocating 1~2 day old nude mice, place them in a Petri dish, wash the nude mice with 1% iodine polyvinylpyrrolidone, rinse thoroughly with running water, and then wash twice with 70% ethanol. Mice should be used immediately. If the number of mice to be treated is large, they can be placed on ice for 30 min.
2. Remove the limbs and tail of the mouse.
3. Make a simple incision from the tail to the skin of the nose. Use the rat's teeth to gently peel off the entire body of the skin, use a pair of tweezers to hold the body, and another tweezers to open the skin.
4. Place the peeled skin tissue on the surface of a sterile petri dish placed on ice until all skin has been collected.
5. Clamp the skin tissue with 2 rat teeth and rinse the skin tissue (the dermis tissue below) in a 2.5% trypsin-containing HBSS sterile Petri dish, then set at 4oC overnight. The epidermis should not be submerged in trypsin solution.
6. Remove the tissue from the trypsin treatment and place the epidermis face down on the surface of a dry sterile cell culture dish. The epidermis is easy to attach to the plastic, and the dermis tissue is pulled off by the tissue clamp. The age of the mouse is affected by this. The older the age, the more difficult it is to remove the epidermis.
7. Carefully cut the residue of the epidermis with scissors and place in a "conventional" medium (MEM containing 10% FCS, 100 IU/ml penicillin, 100 ug/ml streptomycin, 2~4 mmol/L L-glutamine) ; 5 mice per 10 mice in a sterile flask, vigorously stirred at 37 ° C for 45 min in a magnetic stirrer.
8. Filter the liquid with 4 layers of sterile Nitex gauze (16 um, supplied by Martin). The stratum corneum cells remain on the surface of the gauze as other cells pass.
9. Wash the cells with a culture flask. The amount of the medium is about 10 ml of a skin. Inoculate the cells in a plastic cell culture flask or a glass cover slip and incubate at 37 °C for 4 to 12 hours.
10. After 4~12h, observe the cell population on the surface of the culture dish.
11. Transfer the cells to a low calcium medium and the keratinocytes will begin to multiply. This transfer prevents stratification of the cells. The low calcium solution consists of MEM (no calcium) and 10% chelated FCS (also containing normal levels of penicillin, streptomycin). Chelated serum was prepared as follows: 20 g of resin per 50 ml of serum, Chelex 100 (Bio-Rad). The resin was placed in water at 40 g/L and the pH was about 7.4. The filter paper was placed on a funnel and the resin was filtered. Resin was added to the FCS and stirred for 60 min. The serum was then filtered to clarify and sterilized with a 0.45 ug filter paper. The latter filtering process is slower. A commercially available filter is required. The concentration of calcium ions in the complete culture solution was about 0.09 mmol/L, which is a common culture solution compared to the calcium concentration of 0.15 mmol/L.

Precautions

Mouse keratinocytes were maintained in low calcium for up to 1 week, although cell viability was reduced. Keratinocytes can be cultured in low calcium, serum-free medium by commercial means such as Clonetics and GIBCO/BRL. Glial cells can be stored in this culture for a long period of time and in many cases undergo several divisions. In order to induce keratinocyte stratification, normal levels of calcium should be added to the cells.
It is best to use disposable plastic tissue culture equipment instead of glass equipment. If you really need to use glass equipment, apply acid soak and then autoclave. The equipment to be used is also the uncle's dental forceps, pointed anatomical scissors, and bio-absorbent. The above equipment should be autoclaved or soaked and cauterized in 95% ethanol.

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