Cell culture method - Database & Sql Blog Articles

EL-C1600N100013-B

微盟ME2188A33PG

Kaixin micro test ESD18VB03-0201

Brand AVX TPSE226M035R0125 Low impedance tantalum capacitor AVX 22

Materials and reagents
1. Cell: adherent cell line
2. Reagents: 0.25% trypsin, 1640 medium (containing 10% calf serum)
3. Instruments and equipment: inverted microscope, incubator, culture flask, straw, waste tank and other principle cells are basically saturated after the culture flask grows into a dense monolayer, in order to enable the cells to continue to grow, and also the number of cells To expand, it is necessary to pass on (re-cultivate). Subculture is also a way to preserve cell types. It is also a necessary process for culturing cells for various experiments. Suspension cells can be dispensed directly, while adherent cells need to be digested before they can be divided.
Primary tissue block culture
1. Shearing: Place the small pieces of tissue in a small beaker or penicillin vial, rinse with Hanks solution two or three times to remove surface blood stains, absorb the Hanks solution, and cut 1 mm3 pieces repeatedly with an ophthalmic scissors.
2. At the same time: use the elbow pipette to suck a few small pieces, put them in the culture bottle, and use the pipette elbow to place the small pieces of tissue on the bottom of the culture bottle. The distance between the small pieces is 0.5cm, and the bottom of each 25ml culture bottle can be used. At 20 to 30 blocks.
3. Gently flip the culture bottle, and the bottom of the bottle is up. Pay attention to the flow of the small pieces when the bottle is turned over. Plug the stopper and place it in a 36.5 °C incubator for 2 hours (do not exceed 4 hours) to make the small pieces dry. .
4. Culture: remove the culture flask from the micro-cartridge, open the stopper, hold the culture flask at 46 degrees, gently inject the culture solution into the bottom of the bottle, and then slowly turn the culture bottle over, let the culture solution slowly cover. A small piece of tissue attached to the bottle. Static culture in a thermostat. After the number of cells swimming out of the tissue block increases. Add the culture solution.
Subculture method
1. Aspirate the old culture solution in the culture flask.
2. Add a small amount of trypsin and EDTA mixture to the bottle to limit the bottom of the bottle.
3. Place the incubator for 2~5 minutes. When the cytoplasm retracts and the intercellular space increases, the digestion is terminated immediately.
4. Aspirate the digestive juice, inject a few milliliters of Hanks into the bottle, gently rotate the flask and wash away the residual digest. Note that when the cells are washed with Hanks liquid, the action should be light, so as not to wash away the loose cells, such as digesting with trypsin solution, after aspirating the trypsin solution, it can be directly added to the culture solution without washing with Hanks liquid.
5. Drain the nutrient solution with a pipette and gently blow the bottle wall cells to separate them from the bottle wall to form a cell suspension.
6. After the counting plate is counted, the cell suspension is divided into aliquots and placed in several culture flasks, and cultured in an incubator.
Aseptic handling precautions
1. Wash hands before operation. After entering the clean bench, use 75% alcohol or 0.2% Xinjieer to wipe the test. The reagent bottle should also be wiped.
2. Ignite the alcohol lamp, the operation is carried out near the flame. The heat-resistant articles should always be burned on the flame. The burning time of the metal equipment should not be too long, so as to avoid annealing, and the tissue can be picked up after cooling. The utensils that have absorbed the nutrient solution can no longer be cauterized. In order to avoid charring to form a carbon film.
3. The operation should be accurate and agile, but not too fast, in order to prevent air flow and increase pollution opportunities.
4. Do not touch the working part of the sterilized utensils by hand. The layout of the countertops should be reasonable.
5. Try to keep the 45 °C oblique position after the bottle is opened.
6. The pipette of the suction solution cannot be mixed.

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