Preparation of feeder cells (Feeder)
Note: DMEM serum medium (10% FBS) containing 10 ug/ml mitomycin C (MMM 10 mg/mL for laboratory use, Calbiochem)
1. Discard the culture medium in the cell culture dish, add appropriate medium containing 10ug/ml mitomycin C (DMEM + 10% FBS); Shanghai Chuangsai Technology provides mitomycin C, Mitomycin C, ≥ 970 Gg/mg, USP31, BR, can be used for cell culture, 50-07-7, commodity number: C84-1164-5mg, price 259 yuan.
2. Incubate in an incubator for 3 hours (2-4 hours);
3. Treat the culture dish with 0.1% gelatin (add gelatin, shake the gelatin to cover the bottom of the dish, then discard the gelatin and discard it), leave it at room temperature for more than 2 hours, until the gelatin at the bottom of the dish is dry; Shanghai Chuangsai Technology provides gelatin. , Gelatin, product number: B11000719-100g, the price is 241.5 yuan.
4. Discard the medium containing mitomycin C, add 2-3 mL of PBS, shake gently, discard PBS, and wash 3-5 times (must wash more than 3 times in order to thoroughly wash away the remaining mites) Mycin, mitomycin is a mitotic inhibitor and is therefore toxic to ES cells);
5. Add appropriate amount of trypsin for 30 seconds, aspirate the digestive juice, and let it stand until the cell layer has cracks or obvious sliding wall. Add the medium, blow it, and plant it in the gelatin-treated dish (if the cells are too thin) It can be supplemented with mitomycin-treated cells. It can be used after half an hour. (If it is urgent, it can be digested with ES cell culture medium and then transferred to gelatin-treated new plate together with ES cells) It can be used for 6-10 days. Change the culture solution before use. (If the culture dish is not treated with gelatin, it should be placed in the incubator for 2 hours to be used above; it is best to make the Feeder the afternoon before, and use it the next morning. At this time, the Feeder state is the best, and it is most suitable for the growth of ES cells. If the Feeder made the problem found the next morning, it can be done quickly.) Shanghai Chuangsai Technology Co., Ltd. provides 8049-47-6, pancreatin (11×USP), Pancreatin-11.0 from hog pancreas, product number: C84-0033-100g, and the price is 364.8 yuan.
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