Keeping cells, pay attention to these details!(2)
Selection of frozen medium
Cold storage medium must be selected when the frozen cell is stored, and cryopprotectants such as DMSO or glycerin should be included in the cold storage medium.
It can also be used to make a fully frozen medium, such as HyClone, Gibco's cell freeze medium.
The setting of cell culture temperature
The optimal temperature of cell culture depends mainly on the body temperature of the cell donor, and it is also affected by the differences in body temperature of the plane. For example, the skin temperature is lower than that of skeletal muscle.
A more serious problem in cell culture than in low temperature: therefore, the incubator temperature is often set to slightly below the optimal temperature.
Optimum temperature for all types of cell culture
Most of the people and mammalian cell line under 36 ℃ ~ 37 ℃ with the best growth condition.
Insect cells in 27 ℃ with the best growth condition;
In less than 27 ℃ and 27 to 30 ℃ range, the cells grew more slowly.
Temperature exceeds 30 ℃, insect cell activity decreased, even if the temperature drops to 27 ℃, the cell vitality could not recover.
Poultry cell line at 38.5 ℃ with the best growth condition.
Although such cells can also be used in 37 ℃ under cultivation, but its growth speed will slow down.
Is derived from the cold-blooded animals (e.g., amphibians, cold water fish) cell lines can tolerate a wide temperature range, 15 to 26 ℃.
Note that the culture conditions of each cell are different, and that the culture conditions required to deviate from one cell lead to a cell phenotypic abnormality, or even a complete failure.
Therefore, we recommend that you should familiarize yourself with the cell lines that you use, and you will strictly follow the instructions given to all products in the experiment.
The optimal pH of cell culture
Most normal mammalian cell lines can grow well in an environment with a pH of 7.4 and the difference between different cell lines is minimal.
However, some conversion cell lines are found to be better in the mildly acidic environment of pH7.0-7.4, while some normal fibroblasts are more suitable for mildly alkaline environments, namely, the pH of 7.4-7.7.
Insect cell lines such as Sf9 and Sf21 are most suitable for growth in a pH 6.2 environment.
The optimal pH control of cell culture medium
The cell culture medium can control the pH value of the culture system and the pH value of the cultured cells.
This buffer is usually accomplished by adding organic buffer salts (e.g., HEPES) or co2 - bicarbonate buffer salts.
Because the pH of the medium depends on the precise balance between the dissolved carbon dioxide and the bicarbonate, the change in the amount of carbon dioxide in the air will change the pH of the medium.
For this purpose, the use of carbon dioxide - bicarbonate buffer salt medium must be used when exogenous carbon dioxide, especially using open petri dish culture cell or high levels of training in transformed cell lines.
Preservation of serum in cell culture
The serum used in cell culture is not the same, and you need to select the most suitable serum according to the type of cells you cultured and the culture requirements.
We suggest that will be saved in serum - between 10-20 ℃, if stored at 4 ℃, no more than a month.
If you can't use one bottle at a time, it is recommended that you pack the serum aseptically into the appropriate volume of sterilized containers and put them back in the freezer.
When thawed blood serum, it is recommended that you remove the serum from the cooler, after in the first 2-8 ℃ refrigerator overnight melting, and then make all melt at room temperature.
It is important to note that the melting process must be uniformly shaken.
How to avoid the "edge effect" when the cells are laid out
In order to avoid the "edge effect" when the cell experiment was made, the middle 60 hole of the 96-orifice plate was used as the best, and the peripheral perforation of the periphery of the cell was not raised, and only the blank or negative control was done.
When paving, the cell suspension must be mixed to avoid the amount of cells in each hole, which can be mixed every few times.
To be skilled with the sample, try to avoid human error.
Moreover, too many blows can affect cell vitality.
So be familiar with it, and board it as soon as possible.
When to choose to use thermal inactivated serum
The heating serum can inactivate the complement system.
The activated complement is involved in dissolving cell events, stimulating smooth muscle contraction, cell and platelet release histamines, and activation of lymphocytes and macrophages.
When studying immunology, cultivating ES cells, insect cells and smooth muscle cells, it is recommended to use the heat inactivated serum.
Thermally inactivated serum may appear
When studying immunology, cultivating ES cells, insect cells and smooth muscle cells, it is recommended to use the heat inactivated serum.
And for most other cells, there is no need for heat inactivated serum.
Heat inactivated serum for the promotion of the growth of cells only tiny, little or no, even because of high temperature processing affect the quality of the serum, and cause cell growth rate;
Sediment after heat treatment serum, will significantly increase, these deposits observed under inverted microscope, such as "small black spots", often let researchers think serum has been plagued by pollution, and put the serum on 37 degrees in the environment, and makes the sediment increased, the researchers split the amplification of mistaken for microorganisms.
You can choose whether or not to heat up the live serum according to your research.
In this way, you can not only ensure the quality of the serum, but also save your precious time.
How to properly heat and inactivate the serum
When heat inactivated please put serum in 56 ℃ water bath for 30 minutes.
In order to minimize the effect of thermal inactivation on serum quality, an inactivated serum should not be too large.
Best to prepare the same container behave affectedly volume of water, such as (the same as the serum temperature), the containers of serum and water when inactivated in 56 ℃ water bath at the same time, put a thermometer in the water containers, gently rotating blending serum from time to time in the heating process, when the thermometer display has reached around 56 ℃, start the time.
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