Conventional cell culture methods

The first group of tissue culture method

1. Shear: the small pieces of tissue placed in a small beaker or penicillin vials, with Hanks solution rinse two or three times to remove the surface of blood, smoke Hanks solution, with ophthalmic scissors repeatedly cut 1mm3 block so far.

2. Merge: the elbow suction pipe to draw a number of small pieces, placed in the flask, with straw elbow to organize the small pieces at the bottom of the culture bottle, small pieces of distance to 0.5cm is appropriate, each 25ml culture bottle can be About 20 to 30 pieces.

3. gently flip the flask, the other bottle up, pay attention to turn the bottle when no other small pieces of flow, plug the cork, set 36.5 ℃ incubator about 2 hours (not more than 4 hours), so that small pieces dry The

4. culture: from the micro-box out of the culture bottle, open plug, 46 degrees oblique holding the flask, the bottom of the bottom of the box gently injected into the culture medium a little, and then slowly turn the culture bottle over, so that the culture medium A small piece of tissue attached to the bottle. In the incubator. After the cells have increased the number of swells from the tissue block. And then add the culture medium.

Subculture method of the

principle of

cells in the culture bottle into a dense layer, has been basically saturated, so that cells can continue to grow, but

also the number of cells expanded, it must be passaged (re-culture). Subculture is also a way to preserve the cell species. But also the use of cultured cells for a variety of experiments must be through the process. Suspension cells can be directly sub-bottles, and adherent cells need to be digested before the sub-bottle.

Materials and reagents

1. Cells: adherent cell lines

2. Reagents: 0.25% trypsin, 1640 medium (containing 10% calf serum)

3. Instruments and equipment: inverted microscope, incubator, culture flask, straw, waste Cylinder, etc.

Procedure

1. Remove the old culture medium from the culture flask.

2. Add a small amount of trypsin and EDTA to the bottle to fill the bottom of the bottle.

3. Temperature box 2 to 5 minutes, when the cytoplasm retraction, cell gap increases, immediately terminate the digestion.

4. Remove the digestive solution, the bottle into the Hanks liquid number of ml, gently turn the flask, the residual digestion solution washed away. Note that when adding Hanks solution to wash the cells, the action should be light, so as not to loose the cells washed away, such as trypsin solution alone digestion, remove the trypsin solution, can not use Hanks solution rinse directly into the culture medium.

5. Rinse the nutrient solution with a straw to gently blow the bottle wall cells, so that from the bottle wall from the formation of cell suspension.

6. Count the plate count, the cell suspension is divided into equal parts into several culture bottles, incubator in the incubator.

Aseptic Operation Precautions

1. Wash hands before handling, enter the clean bench to use 75% alcohol or 0.2% benzalkonium bromide. Reagents and other bottles have to wipe.

2. ignite the alcohol lamp, the operation in the vicinity of the flame, heat-resistant items should always burn on the flame, metal equipment burning time can not be too long, so as not to annealing, and cooling to clip the organization after the absorption of nutrient solution can not be burning , So as not to form a carbon film charred.

3. Operation to be accurate and agile, but not too fast to prevent air flow, increase the chance of pollution.

4. Can not touch the working part of the sterilized utensils, the table to be reasonable layout.

5. After the bottle opening to try to keep 45 ℃ oblique.

6. Suction solution of straw can not be mixed.

 

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