To grow cells, you also need to pay attention to these details!（2）

Selection of cryopreservation medium

The freezing medium must be selected when freezing cells. The freezing medium should contain cryoprotectants such as DMSO or glycerol.

It is also possible to use specially formulated complete freezing medium, such as HyClone and Gibco’s cell freezing medium.

Setting of cell culture temperature

The optimal temperature for cell culture mainly depends on the body temperature of the cell donor, and is also affected by the difference in body temperature at the unplaning site. For example, the skin temperature is lower than that of the skeletal muscle.

Compared with the temperature that is too low, the problem is more serious when the temperature is too high during cell culture: therefore, the temperature of the incubator is often set slightly lower than the optimal temperature.

The best temperature for all kinds of cell culture

Most human and mammalian cell lines have optimal growth conditions at 36°C to 37°C.

Insect cells have the best growth state at 27°C; cell growth is slower at lower than 27°C and within the range of 27 to 30°C.

When the temperature exceeds 30°C, the insect cell viability decreases, even if the temperature drops to 27°C, the cell viability cannot be restored.

The avian cell line has the best growth state at 38.5°C. Although such cells can also be cultured at 37°C, their growth rate will slow down.

Cell lines derived from cold-blooded animals (such as amphibians, cold-water fish) can tolerate a wide temperature range of 15-26°C.

Please note that the culture conditions for each type of cell are different. Deviations from the culture conditions required for a certain cell can cause abnormal cell phenotypes and even complete culture failure.

Therefore, we suggest that you should be familiar with the cell line you are using, and strictly follow the operating instructions attached to all products in the experiment.

Optimal pH for cell culture

Most normal mammalian cell lines can grow well in an environment with a pH of 7.4, and there are very few differences between different cell lines.

However, it has been found that some transformed cell lines grow better in a mildly acidic environment, that is, at pH 7.0-7.4, and some normal fibroblast cell lines are more suitable for a mildly alkaline environment, that is, pH is 7.4-7.7 .

Insect cell lines such as Sf9 and Sf21 are most suitable for growth in an environment with a pH of 6.2.

Optimal pH control of cell culture media

The cell culture medium can control the pH value of the culture system and buffer the changes of pH value for the cultured cells.

This buffering effect is usually achieved by adding organic buffer salts (for example: HEPES) or carbon dioxide-bicarbonate buffer salts.

Since the pH of the medium depends on the precise balance between dissolved carbon dioxide and bicarbonate, changes in the carbon dioxide content in the air will change the pH of the medium.

For this reason, exogenous carbon dioxide must be used when using carbon dioxide-bicarbonate buffered salt medium, especially when cells are cultured in open petri dishes or high-concentration transformed cell lines are cultured.

Preservation of serum in cell culture

The serum used in cell culture is not the same. You need to select the most suitable serum according to the type of cells you are culturing and the culture requirements.

We recommend storing the serum at -10 to -20°C. If it is stored at 4°C, do not exceed one month.

If you cannot use up one bottle at a time, it is recommended that you aseptically distribute the serum into sterile containers of suitable volume, and then put them back into the freezer for storage.

When thawing the serum, it is recommended that you take the serum out of the freezer, thaw it in a refrigerator at 2-8°C overnight, and then let it completely thaw at room temperature. It should be noted that during the melting process, it must be shaken regularly and evenly.

How to avoid “edge effects” when plating cells

In order to avoid “edge effects” when plating cells in experiments, it is best to use the middle 60 wells of a 96-well plate. Generally, a circle of edge holes on the periphery does not support cells, and only blank or negative controls are used.

When plating the plate, the cell suspension must be mixed to avoid the precipitation of the cells and the number of cells in each well is not equal. You can mix it every time a few are connected.

The operation of the sampler should be proficient and try to avoid human error. In addition, too many blows will affect cell viability. Therefore, it must be proficient in operation and board as soon as possible.

When to choose to use heat-inactivated serum

Heating serum can inactivate the complement system.

Initiated complement participates in cell lysis events, stimulates smooth muscle contraction, cells and platelets release histamine, and initiates activation of lymphocytes and macrophages.

In immunological research and culture of ES cells, insect cells and smooth muscle cells, heat-inactivated serum is recommended.

Possible phenomena in heat-inactivated serum

In immunological research and culture of ES cells, insect cells and smooth muscle cells, heat-inactivated serum is recommended.

For most other cells, heat-inactivated serum is not required.

Heat-inactivated serum only slightly promotes the growth of cells, or has no effect at all. It may even reduce the cell growth rate due to the high temperature treatment affecting the quality of the serum; after heat treatment, the sediment will increase significantly. Observed under an inverted microscope, these precipitates are like “little black spots”, which often make researchers mistakenly believe that the serum is contaminated, and putting the serum in a 37°C environment will increase the amount of precipitates, causing researchers Misunderstood as the division and expansion of microorganisms.

You can choose whether you need heat-inactivated serum according to your research needs.

In this way, not only can the quality of the serum be ensured, but also your precious time can be saved.

How to properly heat-inactivated serum

When heat inactivating, put the serum in a 56℃ water bath for 30 minutes.

In order to minimize the effect of heat inactivation on the quality of serum, the volume of serum to be inactivated at one time should not be too large.

It is best to prepare the same container with the same volume of water (at the same temperature as the serum). When inactivating, put the container with serum and water into a 56°C water bath at the same time, and place a thermometer in the container with water. Rotate gently to mix the serum. When the thermometer shows that it reaches about 56°C, start timing.