1. How to store and thaw serum without impairing product quality?
After the serum was taken out of the freezer, it was first placed in a refrigerator at 2 to 8 ° C to be melted, and then completely melted at room temperature. However, it must be noted that the melting process must be uniformly shaken regularly. When stored at 2-8 ° C for a long time, various proteins and lipoproteins (such as cold agglutinin, fibrinogen, villogen, etc.) in the serum may aggregate to form a precipitate or visible turbidity. Therefore, it is recommended to store serum below -20 °C and avoid repeated freezing and thawing. We recommend that serum should be stored at -2O °C. Do not exceed one month when stored at 4 °C. If it is not possible to use one bottle at a time, it is recommended to sterilize the serum into a suitable sterilization container (since the serum will increase by about 10% when frozen, the volume of this expansion volume must be reserved, otherwise it is prone to contamination or container freezing. In the case of cracking, put it back in the frozen.
2. The serum contains flocculent precipitates. What are they and how to deal with them?
The precipitate contains fibrin and lipoproteins. This is a normal feature and does not affect the nature of the product. To remove the pellet, centrifuge the serum (400g, 1-2 minutes) or simply allow it to settle on the bottom of the bottle and carefully transfer the serum to another sterile vial (generally it is not recommended to remove the precipitate by filtration because precipitation will Block the filter and not filter). In most cases, the precipitate is gently shaken and heated to 37 ° C to dissolve. Therefore, when using the product, shake and heat the serum to 37 ° C, the precipitation will naturally disappear.
3. How to avoid precipitation in serum?
Precipitation can be avoided by doing the following:
(1) When thawing serum, please shake it at any time to make the temperature and composition uniform, and reduce the occurrence of sedimentation.
(2) When cryopreservation of serum, it should be shaken regularly (be careful not to cause bubbles) to make the temperature and composition uniform.
(3) Do not directly thaw from -20 °C to 37 °C. Because the temperature changes too much, it is easy to cause protein condensation and precipitation.
(4) Do not leave the serum at 37 °C for too long, otherwise the serum will become cloudy, and many of the more unstable components in the serum will be destroyed, thus affecting the quality of the serum.
(5) The heat inactivation of serum is very likely to cause an increase in sediment, which may not be necessary if not necessary.
(6) If heat inactivation of serum is necessary, please observe the principle of 56 ° C, 30 minutes, and shake it at any time. If the temperature is too high, the time is too long or the shaking is uneven, it will cause an increase in sediment.
4. After adding serum and antibiotics to the medium, can it be stored for a long time?
Once you have added serum and antibiotics to your fresh medium, you should use it within two to three weeks. Because some antibiotics and essential components in serum begin to degrade after thawing.
5. Why do you want to heat inactivate serum?
Heating can inactivate the complement system. Activated complement is involved in lytic cell events, stimulates smooth muscle contraction, releases histamine from cells and platelets, and activates lymphocytes and macrophages. In immunological studies, it is recommended to use heat-inactivated serum when culturing ES cells and smooth muscle cells.
6. Is it necessary to do heat inactivation?
Experiments have shown that properly treated heat-inactivated serum is not required for most cells. The serum thus treated has only a slight promotion to the fine growth, or has no effect at all, and even the high temperature treatment usually affects the quality of the serum, resulting in a decrease in the cell growth rate. The heat-treated serum, the formation of precipitates will increase significantly, these precipitates observed under an inverted microscope, such as "small black spots", often let the researchers mistakenly believe that the serum is contaminated, and put the serum in 37 In the °C environment, this precipitate will be increased, which makes the researchers mistakenly believe that it is the splitting and amplification of microorganisms. If not necessary, you do not need to do the heat treatment step. Not only save time, but also ensure the quality of serum!
7. Is black spot in cell culture polluted? How to deal with it?
Once you have found black spots in the cell culture process, first, visually observe whether the medium is turbid, and then observe the state of the cultured cells under the microscope, and whether the black spots are swimming. If the cells are contaminated, the microorganisms will multiply and the medium will turn yellow and become cloudy. Contamination includes bacterial contamination, mycoplasma contamination, and the like. If the cells are observed under the microscope, the growth state is good, and there is no change compared with before the black spots appear. Then, the appearance of black spots may be related to the following situations:
(1) cells grow too old, broken cell debris;
(2) The serum quality is not good, and the results of repeated freezing and thawing;
(3) The pH of the preparation medium is too high, and cell growth is not suitable;
(4) The water quality and container of the prepared medium are unqualified. Centrifugal, filtration methods can be used to remove these black spots;
(5) Small black spots appear in the cultured primary cells, which may be impurities in the primary tissues, which can be eliminated by multiple passages.
8. What are the differences and precautions between calf serum and fetal bovine serum?
Different sources: Fetal Bovine Serum (FBS) is a fetal bovine serum obtained by blood sampling by cardiac puncture when slaughtering pregnant cows. Newborn calf serum (CS) is collected from birth 10 to 14 days of calf. The composition is different from the ratio: the components of the growth-promoting factor, the adhesion-promoting factor, the hormone and other active substances are different from each other, and the use and usage are different: some cells must have fetal bovine serum to grow, and some The cells only need calf serum, and the concentration is different, generally 5%-10%, and the concentration is 20%.
Serum storage and use should be noted: serum should be stored at -5 ° C to -20 ° C, if stored at 4 ° C can not exceed one month. When thawing serum, the serum should be placed in a refrigerator at 2-8 ° C, and shaken frequently to dissolve it, then placed at room temperature to warm it up. Never put the frozen serum directly into a 37 ° C water bath or incubator, such as Thaw at 37 ° C, the color is deepened, the viscosity will also increase. After thawing and heat inactivation, the serum should be stored according to the dosage and stored at -20 ° C to avoid repeated freezing and thawing.
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