Primary cultures of mouse hepatocytes can be used (1) for cell preservation, (2) for molecular biology research, and (3) for gene therapy research.
Operation method
trypsin digestion
Principle
Hepatocytes from mice are removed from the organism, treated with trypsin, chelating agent (commonly used EDTA), dispersed into single cells, and cultured in a suitable medium to allow the cells to survive, grow, and multiply.
Materials and Instruments
Mouse Move 1. The mice were killed by breaking their necks, placed in a 75% alcohol bath for 2-3 seconds, and the livers were removed and placed in a flat dish containing PBS. 2. Remove fat, connective tissue, blood and other debris and transfer to another flat dish with PBS solution. 3. Using surgical scissors, cut the organ into small pieces (approximately 1 mm2 in size), grind the slide, transfer to a centrifuge tube, and centrifuge (1 000 rpm, 5 min). 4. Depending on the amount of tissue or cells, add 5-6 times (3-5 ml) trypsin and digest for 20 min at 37°C, shaking every 5 min or blowing with a pipette to separate the cells. 5. Add 3-5 ml of culture medium containing serum to abort trypsin digestion. 6. Strain through a 100 mesh screen to remove large undigested tissue mass. 7. Centrifuge again for 5 min and discard the supernatant. 8. 8. Add 5 ml of serum-free culture medium to disperse the cells, centrifuge again, and discard the supernatant. 9. Add 1-2 ml of serum-containing culture medium (depending on the amount of cells) and count the cells on a hemocyte counting plate. 10. Adjust the cells to about 5×105/ml, transfer to a 6-well culture plate and incubate at 37℃. Caveat 1. Maintain all tissue cells in sterile conditions from the time of sampling. Cell counting may be performed in a sterile environment. 2. In the ultra-clean table, tissue cells, culture solutions, etc. should not be exposed for too long to avoid evaporation of solutions. 3. where steps are operated outside the ultra-clean bench, each vessel needs to be covered with a lid or rubber stopper to prevent bacteria from falling in. 4. Wash your hands before operation, and wipe your hands with 75% alcohol or 0.2% Neosporin after entering the ultra-clean bench. The mouth of the reagent bottle should also be wiped. 5. Light the alcohol lamp, operate near the flame, heat-resistant items should always be burned on the flame. Metal instruments should not be cauterized for too long to avoid annealing and should be cooled before clamping the tissue. Appliances that have absorbed nutrient solution should not be cauterized again to avoid charring and forming a carbon film. 6. the operation should be precise and agile, but not too fast to prevent air flow, increasing the chance of contamination. 7. the working part of the sterilized utensils should not be touched by hand, and the arrangement of supplies on the workbench should be well laid out. 8. bottles should be kept in a 45° slanting position as far as possible after opening. 9. Pipettes, etc., for sucking up solutions should not be mixed. For more product details, please visit Aladdin Scientific website.
DMEM Serum-free DMEM medium Trypsin PBS
Lunch box Gauze Scissors Tweezers Beakers Flatware Grinding slides Strainers Centrifuge tubes 6-well plates Straws Pipettes Gloves Microsampler