V. culture of cell culture
1. When cells grow to high density, they should be divided into new culture bottles. The general dilution ratio is 1:3 to 1:6, depending on the type of cell.
2.1. The pancreatic enzyme solution (0.05% trypsin) : 10 ml packing in 15 ml sterile centrifuge tube, preserved in 20 ℃, in 37 ℃ water tank before use.
2.3. Fresh culture medium 2.4. Sterile straws/centrifuges/cultured bottles
3.1. Adhesion cell (adherentcell)
3.1.1. Remove the old medium.
3.1.2. Wash cells with d-pbs for one to two times.
3.1.3. Join the pancreatic enzyme solution (1 ml / 25 cm2, 2 ml / 75 cm2), 37 ℃ for a few minutes, in inverted microscope, when which appear round granular cells is, suck or dumping of pancreatic enzyme solution.
(if not completely removed, after pancreases, add the right amount of fresh culture medium containing serum to terminate the pancreatic enzyme)
3.1.4. Pat culture bottle cell falls off the bottle wall, adding suitable amount of fresh medium, to straw to suck up and down several times to break cell mass, evenly mixed, and transferred to the new culture in a bottle, in proportion to dilute with normal cultivation conditions.
3.2.1. Take out the cell culture medium and put it in the centrifuge tube, and centrifuge 1000rpm5-10 minutes.
3.2.2. Remove the supernatant liquid and add the appropriate amount of fresh culture medium. After mixing and homogenize, transfer to the new culture bottle according to the dilution ratio, and cultivate the normal culture condition.
3.3. Hybridioma cells
Some hybrids need to be trained for more than three days to produce antibodies. If they change the medium, they may lose their antibodies.
Therefore, the secondary culture should not be replaced by the culture medium, and the concentration can be added directly to the new culture medium.
If the volume is too large, it can be tilted or placed in a new culture bottle.
Vi. Cell cryopreservation
1.1. The cells that are intended to be cryopreservation should be in a well - growing index with a high survival rate of about 80-90%.
1.2. Whether the cell retains its unique properties before freezing, for example, the cell of the hybridioma should be tested one to two days prior to freezing
Try to produce antibodies.
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1.3. Note the quality of cryoprotectants.
DMSO should be reagent grade, sterile and colorless.
4 ℃ avoid light preservation, do not make multiple thaw.
Glycerol should also be used as reagent grade to avoid light preservation after high pressure steam sterilization.
It is used within one year after opening and is toxic to cells after long-term storage.
1.4. Cryopreservation cell concentration: www.bbioo.com
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1.4.1. Normal human fibroblasts: 1-3 x 106 cells/ml
1.4.2. Miscellaneous tumor cells: 1-3x106cells/ml, the cell concentration is not too high, and certain hybridioma cells will die after thawing 24 hours after the freezing concentration is too high.
1.4.3. Tumor cell lines of pasted wall: 5-7x106cells/ml, depending on the type of cell.
The concentration of adenocarcinoma should be higher after thawing, while HeLa only needs 1-3 x 106cells/ml.
1.4.4. Other suspension cells: 5-10 x 106 cells/ml, humanlymphocyte must be at least 5 x 106 cells/ml.
1.5. The concentration of refrigerant is 5% or 10% DMSO, if the freezing condition of the cell is not determined, it is the same as freezing
A control culture should also be made to prevent freezing failure.
2. Specific operation of cell cryopreservation
2.1. Cultured cells of good growth
2.2. Fresh medium
2.3. DMSO (SigmaD - 2650)
2.4. Sterile plastic cryopreservation tube
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2.6. Blood count plates and cover slides
3.1. Replace the half-dose or total medium before freezing, and observe the growth of cells.
3.2. Preparation of cryopreservation solution (pre-made) : add DMSO to the fresh medium, and the final concentration is 5-10%.
Mix well and place at room temperature.
3.3. The cultured cells were collected in accordance with the operation of the cell culture, and a small amount of cell suspension (about 20ul) was collected
Degree and pre-freeze survival.
3.4. Centrifugal, remove the supernatant, adding suitable amount of cryopreservation solution, the corresponding cells suitable density of cryopreserved mixing, packing in marked the cryopreservation of tube completely, 1 ml/tube, and take a small amount of cell suspension for pollution detection.
3.5. Cryopreservation method: indicate the name of the cell line on each frozen storage tube, the name of the cryopreservation and the storage time of the cryopreservation, which is convenient for future search.
To bind a group of cells with a rubber band, with enough cotton will be cryopreserved tubes, including the base of the beaker and bottle top, protect cells by cooling, prevent rapid frozen state of ice damage cells, into the frozen - 80 ℃, and then placed in liquid nitrogen.
vii. Freeze cell activation
1. The activation principle of frozen cells is to defrost rapidly, so as to avoid the damage caused by the recrystallization of ice crystals, resulting in cell death.
2. After cell activation, it takes about a few days, or a second or second generation, and its cell growth or characteristic will return to normal (for example, production
To produce monoclonal antibodies or other proteins.
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3. The material of 37 ℃ constant temperature water, fresh medium, germ-free straw/centrifugal tube/bottle, liquid nitrogen or carbon dioxide ice
4.1 the operator shall wear protective mask and gloves to prevent the damage of the tube from bursting.
4.2 remove the frozen tube from the liquid nitrogen or dry ice container, check whether the lid is tightness, because the heat expansion and shrinkage process, the lid is easy
4.3 put fresh medium at 37 ° C in the sink back to wen, rebound after spraying with 70% alcohol and wipe it, moved to the bench.
4.4 remove the frozen pipe immediately in 37 ° C rapid thawing in the sink, rocking freeze pipe to make it melt in 1 minutes with 70% ethanol to wipe save tube outside, to the bench.
4.5 remove the cell suspension of 0.9ml of thawing and slowly add to the culture medium (dilution ratio 1:10 ~ 1:15) and mix evenly, put in the CO2 culture box culture.
A 0.1ml unthawed cell suspension was used for survival test.
4.6 if thawed immediately remove cryoprotectants (such as DMSO or glycerol), according to the types of cells and, in general most don't need to immediately remove cryoprotectant.
But if you want to remove immediately, will release the cell suspension of joined the centrifugal tube containing 5-10 ml medium, centrifugal 1000 RPM, 5 minutes, remove the supernatant, add fresh medium, mixing, into the CO2 incubator culture.
4.7 if the refrigerating preservatives are not removed immediately, the media should be changed the next day after thawing.