Cell Culture FAQ

1. How to determine whether a cell is contaminated by mycoplasma?

Direct culture method, fluorescence staining or PCR method can be chose to detect contaminants. The more commonly used method is PCR. When the cells are contaminated by mycoplasma, in principle, the mycoplasma can be removed, but the whole process is time-consuming and labor-intensive. Therefore, if it is not in a situation where the cell stock is very small, it is recommended to discard it immediately and re-cultivation when it is found to be contaminated.

2. What is the best way to preserve serum?

It is recommended that the serum should be stored at -5°C to -20°C. However, if stored at 4°C, do not exceed one month. If it is not possible to use up one bottle at a time, it is recommended to aseptically distribute the serum into an appropriate sterilized container, and then put it back into the freezer.

3. How to thaw the serum without impairing its quality?

It is recommended that put the serum in a refrigerator at 2～8℃ to melt it after taking it out of the freezer. And then melt it completely at room temperature. However, it must be noted that during the melting process, it must be shaken regularly and evenly.

4. What to do if there is flocculent sediment after the serum is thawed?

There are many reasons for the appearance of sediment in the serum, but the most common cause is the denaturation of lipoproteins in the serum. Fibrin (one of the proteins that form clotting) will exist in the serum after the serum is thawed, which is also one of the main causes of deposits.

But these flocculent sediments do not affect the quality of the serum itself. If you want to remove these flocculent sediments, you can aliquot the serum into a sterile centrifuge tube, centrifuge it slightly at 400 g. Then add the supernatant to the culture medium and filter them together. We do not recommend that you use filtration to remove these flocculent sediments, because it may clog your filter membrane.

5. How to control the time of trypsin digestion?

The degree of trypsin digestion is a key point in cell culture. Excessively digested leads to cell debris and black scum increasing. Cells will fall off in pieces, which seriously affects cell viability. Some cells float and lose with the discarded trypsin. Insufficient digestion results in the difficulty of cells to be blown down from the bottle wall, and repeated pipetting also damage cell viability.

The sensitivity of different cells to the digestion fluid is different, and the time of trypsin digestion is also different. The digestion time is related to the concentration of trypsin, whether it contains EDTA, the storage time and temperature of trypsin, whether it is repeatedly freeze-thaw, the volume of trypsin added, the digestion temperature and the density of cells. Digestion for newly purchased cells, it is recommended that customers can use low concentration gradient trypsin to carefully explore the digestion time. Observe whether the cells are rounded every 1 minute and record the best digestion time.

6. Why use trypsin-EDTA solution?

EDTA is used to chelate free magnesium ions and calcium ions in order to maintain the activity of inhibiting trypsin. It is recommended to wash the cells with EDTA before trypsinizing the cells to eliminate all divalent ions from the culture medium.

7. Can you use a serum type that is different from the original one?

The serum is an extremely important source of nutrients for cell culture, so the type and quality of serum have a great impact on cell growth. Serum from different species differs in the amount or content of some substances or molecules, and incorrect use of serum often results in the inability of cells to survive.

8. How to keep the CO2 incubator clean?

Change the water in the water tray regularly (once a week). The water in it must be sterile distilled water or deionized water. 1% copper sulfate can be added to prevent mold contamination.

After the CO2 incubator is contaminated by mold, all cells should be temporarily transferred. Scrub the incubator (including the partition and the wall) with peracetic acid. Put the peracetic acid in the incubator for one hour to diffuse the steam. After the smell of peracetic acid has dissipated, move it into the cells.

9. Some black spots often appear during cultivation. Is it pollution? How to prevent and deal with it?

9.1 Judgment method

Firstly, observe whether the culture medium becomes turbid by naked eyes. If it becomes turbid, it can be basically confirmed as contamination. If not, observe under the microscope whether the size and shape of the black spots are regular, whether they are moving, whether they are doing Brownian motion or are in a straight line type fast-moving. If the black dots have the irregular size and perform Brownian motion, they may be cell debris (poor cell condition or over-digestion). Protein precipitation after repeated freezing and thawing of serum, as well as cellular metabolites, may also form black spots. If the black spots are the same size and move quickly, it is likely to be bacterial contamination.

9.2 Preventive measures
1) Grasp the best time for cell passage, instead of when it grows old.
2) Master the digestion time to prevent cell debris from being over-digested.
3) Reduce the repeated freezing and thawing of serum and other reagents. Adjust the pH of the culture medium to the best.
4) Strictly control the cleaning of water and utensils.

9.3 Treatment method

If it is determined that the black spots are contaminants, please dispose of the cells in time and discard them. In other cases, please refer to the following methods.

For suspension cells

Collect the cell supernatant by slow centrifugation (500-600 rpm/min, 5-6 min) and replace with a new culture flask.

For adherent cells

Wash the cells 2-3 times with PBS. When washing, gently tap the culture flask to remove the fragments and particles that are not firmly attached. Then discard the PBS and add low-concentration trypsin(e.g. 0.05%, 1 min) to digest, allowing particles and debris in the intercellular space to fall off. Remove low-concentration trypsin, and then digest the cells normally. Centrifuge the collected cell suspension at a slow speed (500-600rpm/min, 5 -6 min) and replace the culture flask with a new one. Increase the serum concentration appropriately for culture.

10. How to distinguish live cells from dead cells? How to count live cells with trypan blue?

Observe under the microscope. The living cells are bright, full and shiny, while the dead cells are darker. Cell viability can be calculated by staining with trypan blue.

Dilute the cell suspension to 200-2000 cells/ml with serum-free medium, and add 0.1 ml 0.4% trypan blue solution to 0.1 ml cell suspension. Mix gently and count the cells with a hemocytometer. Live cells reject trypan blue, so the cells stained blue are dead cells. Note that the count should be completed within 10 minutes after adding trypan blue. If there is a cell counter, it can be performed directly with a counter.