In the cell culture laboratory, during the process of cell culture, some problems may arise, such as how to quickly thaw the cryotubes, when to change the culture medium, and whether to use 5% or 10% CO2 during cell culture. The editor has summarized these problems and answered them one by one.

What are FBS, FCS, CS and HS?

FBS (fetal bovine serum) and FCS (fetal calf serum) have the same meaning. Both refer to fetal bovine serum. FCS is an incorrect usage. Please do not use it anymore. CS (calf serum) refers to calf serum. HS (horseserum) refers to horseserum.

2. How should the freezing tube be thawed?

After taking out the freezing tube, it must be immediately placed in a 37 ℃ water tank for rapid thawing. Gently shake the freezing tube to make it completely melt within one minute. Also, be sure that the water level does not exceed the edge of the freezing tube cover, otherwise contamination is likely to occur. When the freezing tubes are taken out of the liquid nitrogen tank for thawing, safety must be observed to prevent the tubes from bursting.

When thawing and culturing cell cryotubes, should DMSO be removed immediately?

Except for a few cells specifically noted as sensitive to DMSO, the vast majority of cell lines (including suspension cells) should be directly placed in a culture flask containing 10-15 mL of fresh medium after thawing. The next day, the fresh medium should be replaced to remove DMSO. This can avoid the problem that most cells cannot grow or adhere after thawing.

4. When should the culture medium be replaced?

It depends on the growth density of the cells or follows the replacement time indicated on the basic data of the cell line. Just change the culture medium on time.

5. Is it necessary to add antibiotics to the culture medium?

Except for special screening systems, no antibiotics should be added to the culture medium under normal culture conditions.

6. Can a culture medium with different conditions from the original one be used?

No. Each cell line has its specific cell culture medium that has been used and adapted to. If a culture medium with different conditions from the one originally provided is suddenly used, most cells cannot adapt immediately, resulting in their inability to survive.

7. Can serum types that are different from the original culture conditions be used?

No. Serum is an extremely important source of nutrition in cell culture, so the type and quality of serum have a significant impact on the growth of cells. Serum from different species varies in the amount or contents of some substances or molecules. Incorrect use of serum often leads to the failure of cells to survive.

When culturing cells, should 5% or 10% CO2 be used?

In general culture media, HCO3-/CO32-/H+ is mostly used as a pH buffering system, and the content of NaHCO3 in the culture medium will determine the concentration of CO2 that should be used during cell culture. When the content of NaHCO3 in the culture medium is 3.7g per liter, 10% CO2 should be used for cell culture. When the NaHCO3 in the culture medium is 1.5g per liter, cells should be cultured with 5% CO2.

9. What is the concentration of trypsin-EDTA used in the subculture of attached cells? How should it be handled?

The commonly used concentration of trypsin-EDTA is 0.05% trypsin-0.53 mg EDTA.4 Na. After the first opening of the bottle, it should be immediately aliquot in small amounts into sterile test tubes and stored at -20 ℃ to avoid the reduction of trypsin activity caused by repeated freezing and thawing, and to reduce the chance of contamination.

10. How should suspension cells be subcultured?

Generally, it is only necessary to continuously add fresh culture based on the original culture Angle flask to dilute the cell concentration. If there is too much culture medium, the mouth end of the culture Angle flask can be slightly raised until it can no longer hold. When dividing the bottles, take out a portion of the culture medium containing cells and transfer it to a new culture corner flask. Add fresh culture medium and dilute it to an appropriate concentration. Repeat the above steps.