Mouse Hepatocyte Culture Experiment
Mouse Hepatocyte Culture Experiment
This experiment focuses on primary culture of mouse hepatocytes as the first step in establishing various cell lines.
Operation method
Cell Culture Technology
Principle
The first culture of tissue cells obtained directly from an organism is called primary cell culture. Primary cell culture is the first step in establishing various cell lines, and it is the most basic technique that people engaged in culture work should be familiar with and master. Depending on the culture method, it is divided into tissue block culture method and monolayer cell culture method.
Materials and Instruments
Mouse Move I. Experimental steps 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. Common Problems Source Tutorials in Agricultural Biotechnology For more product details, please visit Aladdin Scientific website.
DMEM DMEM Trypsin PBS
Lunch box Gauze Small clippers Small tweezers Large tweezers Large beakers Flatware Grinding slides Strainers Centrifuge Tubes 6-well plates Straws Pipettes Pipettes Gloves Microsampler
1. Cut off the neck of the mouse and put it in 75% alcohol for 2-3 seconds, take the liver and put it in a flat dish with PBS.2. Remove fat, connective tissue, blood and other debris and transfer to another dish containing PBS.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 (1000 rpm, 5 min).4. Add 5-6 times (3-5 ml) trypsin, depending on the amount of tissue or cells, and digest at 37°C for 20 minutes, shaking every 5 minutes 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 strainer to remove large undigested pieces of tissue.7. Centrifuge again for 5 min and discard the supernatant.8. Add 5 ml of serum-free culture medium, 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 with a hemocyte counter plate.10. Adjust the cells to about 5x105/ml, transfer to a 6-well culture plate and incubate at 37℃.