1. When you receive the cell line package, please check the cell line cryotube for thawing, and if so, please notify us immediately. Please start culturing the cell line as soon as possible, or freeze it immediately (at –70 ° C, move to liq N2 overnight).
2. Frozen cell thawing procedure:
2.1. Prepare the culture medium based on the basic medium type, serum type and other specified components and ratios specified in the cell line data sheet. The vast majority of cells cannot immediately adapt to different basic media or different serum types. If they must be different due to experimental needs, it is necessary to gradually change the medium composition at a slow rate to determine the cells after adaptation. Experiment.
2.2. FBS (fetal bovine serum), CS (calf serum) and HS (horse serum) are very different for cells. Please be sure to use the serum specified in the cell line data sheet Species cultivated.
2.3. Put the culture medium in a 37 ° C water tank and return to temperature. After returning to the temperature, spray with 70% alcohol and wipe it, then move it into a sterile operating table. Take out the freezing tube and immediately put it in a 37 ° C water tank for rapid thawing. The height of the water surface should not be close to or higher than the cover of the freezing tube, otherwise it will be easily contaminated. After gently shaking the cryotube to make it melt within 1 minute, wipe the outside of the cryotube with 70% ethanol and move it into a sterile operating table.
2.4. According to the type and concentration of cells, take 10 ml of medium in a sterile operating table and add it to a T25 or T75 flask. Take out the thawed cell suspension, slowly add the medium in T25 or T75 flask, mix well, put in 37 ° C, 5% CO2 incubator for incubation.
2.5. For the vast majority of cells, the cryoprotectant DMSO below 1% will not adversely affect the attachment or activation of cells, and does not need to be immediately removed from the thawed cells. Remove it after attaching it well. However, for the very few cells that are sensitive to DMSO or may cause cell differentiation, and you need to remove DMSO immediately, you can put the thawed cell suspension in 5-10 ml medium and centrifuge at 300 xg (about 1000 rpm). After 5 minutes, carefully remove the supernatant, add an appropriate amount of fresh medium, mix the cells evenly, transfer to a culture bottle, and then put them in a 37 ° C, 5% CO2 incubator.
When receiving T25 flask cells, the processing method is:
1. During the delivery process, in order to avoid foaming and cell death, T25 flasks are filled with medium. Please check the appearance of flask and observe the cell growth and contamination under the microscope. If there is any problem, please do not open the lid, please inform the cell laboratory immediately.
2. Place the intact T25 flask in a 37 ° C, 5% CO2 incubator to warm the cells to 37 ° C, and allow a few cells that have fallen off during transport to attach and grow again. After the next day, remove the culture medium in flask in a sterile operation box (the removed medium can be reused), leaving only about 5-10ml of culture based on flask, and then put the cells into the incubator according to the general culture method Or the cells have grown to full size, then subculture the cells.
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