1. Take 12.5-13.5dpc pregnant rats and sacrifice them by neck;
2. Place the rat belly up, 70% alcohol to moisten, disinfect the abdomen (to prevent the belly hair from flying, pollute the internal organs and uterus), cut the skin layer, muscle layer, open the abdominal cavity, connect the connective tissue and fat shear attached to the uterus Go, remove the uterus and transfer to a sterile 10cm dish;
3. Transfer the plate to the clean bench;
4. Open the uterine wall with tweezers, squeeze out the mouse embryo, and separate it from the extraembryonic tissue, placenta, etc., to remove the head, limbs and various internal organs of the mouse embryo (mainly liver, lung, heart and stomach);
5. Transfer the mouse embryo to another new sterile dish and wash it three times with PBS;
6. Discard the PBS, cut the mouse embryo with a elbow, and add PBS to wash until the solution is basically colorless (1-4 times);
7. The addition of an appropriate amount of Trypsin-EDTA (0.25%), approximately equal to the volume of the volume of tissue mass; Shanghai, a technology to provide race 610769-35-2, Tris Borate EDTA buffer, for molecular biology, 10x, DNAse, RNAse and Protease free, pH 8.3 , Item No.: C82-32733-1LT, the price is 763 yuan.
8. Blow gently with a dropper, digest for 1-10 minutes at room temperature (or digest until the solution becomes cloudy and sticky), add an equal volume of medium to the digestive juice, mix gently by gently blowing (5-10 times), and let stand. ;
9. After settling, gently aspirate the upper solution and transfer to a centrifuge tube.
10. Centrifuge the cell suspension tube (1000 to 5 minutes);
11. Discard the supernatant, add appropriate amount of medium to the pellet, gently hang and resuspend, divide it into 10 cm cell culture dish, add appropriate amount of medium, label (ME-0; Note: if frozen , you can use the 0 generation ME after resuscitation to make the Feeder; if it is used directly, it is better not to use the 0 generation ME cells as the Feeder, it is better to use it after the generation is used to make the Feeder), and then transferred to the incubator for cultivation;
12. Change the cell depending on the growth of the cells (usually change the liquid once every 3 days). If the cells are full, freeze them, or pass them 1:3-5 (depending on the density of the cells), and put them into the incubator to continue the culture ( After that, there is no need to change the liquid); the 1-5 generation ME cells can be used to make the Feeder.
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