experiment equipment
Three small surgical scissors, three ophthalmologists, two ophthalmologists, three sets of glass plates, 200 mesh nylon filters, 50ML, 15ML centrifuge tubes and surgical blades. All of the above items require autoclaving.
Experimental reagent
Ca 2+ and Mg 2+ free PBS, 0.05% trypsin 0.53 mmol/L EDTA solution, mouse embryo fibroblast (MEF) growth medium: high glucose DMEM plus 10% FCS.
Experimental animal
Pregnant mice between 14 and 16 days of pregnancy
Experimental procedure
1. Execute the pregnant rat, soak it in 75% alcohol, then cut the outer skin of the pregnant mouse with a pair of scissors and a pair of tweezers in the clean bench, and cut the endothelium with another pair of scissors and a pair of tweezers to expose In the uterus, the uterus was carefully removed with a third pair of scissors and forceps and placed in a glass dish containing PBS, and rinsed to remove blood.
2. Carefully remove the outer membrane of the embryo with two curved tweezers, then clip the head and internal organs, transfer the remaining embryos to a 50ML centrifuge tube containing 30MLPBS, gently invert twice, pour off the PBS, and repeat Once the procedure was done, the embryos were transferred to another dish containing PBS with a little PBS and shredded with a surgical blade.
3. Using a 200 ul pipette to repeatedly quickly blow the liquid in the plate, place it in a 15 ML centrifuge tube, centrifuge at 1500 rpm for 5 minutes at 4 ° C, pour off the supernatant, add 10 ML trypsin, resuspend the pellet, and digest it in a 37 ° C water bath. For 30 minutes, shake gently every five minutes to fully digest.
4. The upper cell suspension was poured into a 50 ML centrifuge tube containing 10 mLMEF growth medium, filtered through a 200 mesh nylon mesh, and centrifuged at 1500 rpm for 5 minutes to collect the cells. The 30 mL MEF growth medium was washed twice (in this step, the authors tried washing with serum-free medium and the results were indistinguishable).
5. Cell pellet was suspended with 15 ML growth medium, and cell count (2-3 x 10 7 cells were obtained for eight 14-day fetuses).
6.3 x 10 6 cells were suspended in 15 ML MEF growth medium and inoculated into a 200 ML culture flask.
7. Fresh MEF growth medium was replaced after 24.24 hours.
8. After the cells are full, first rinse with PBS, pour off, add trypsin digestion (this step should not be too long, the author generally does not exceed five minutes), pass 1:5 passage.
9. The cells grow to a coverage of 80-90% again, and after they are digested, they are routinely frozen. (Frozen storage solution should be matched)
V. Discussion
1. Primary culture, we must avoid pollution.
2. The survival ability of MEF is limited. If it is not frozen, it will survive for about ten generations in vitro.
3. Cells that have recovered after cryopreservation can only be passaged once because these cells have limited reproductive capacity.
4. Do not digest cells for too long.
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