Gibco fetal bovine serum use method In the cell culture process, 5%-20% Gibco fetal calf serum is often added, and the most commonly used Gibco fetal bovine serum concentration is 10%. High concentrations of serum may alter the gene expression profile of the cells, affecting the results of subsequent experiments. We used 10% Gibco Australian fetal bovine serum to culture 293T cells in the experiment, which was very effective. However, some cells also use 5% Gibco fetal bovine serum or 20% Gibco fetal bovine serum, and the appropriate Gibco fetal bovine serum concentration is selected according to the specific cells.
Gibco fetal bovine serum preservation method 1. Gibco bovine serum that requires long-term storage, such as Gibco South American fetal bovine serum, must be stored in a -20 ° C - 70 ° C low temperature freezer. Do not store in a refrigerator at 4 °C for more than 1 month. Do not leave the serum at 37 °C for too long, otherwise the serum will become cloudy, and the active ingredients in the serum will be destroyed, which will affect the serum quality. If one bottle cannot be used at a time, 40 to 45 ml can be dispensed in a sterile 50 ml centrifuge tube. Since the volume of the serum will increase by about 10% when frozen, the serum must be reserved for a certain volume before it is frozen into the low-temperature refrigerator, otherwise it may be contaminated or the glass bottle may be cracked.
2, the serum provided by the general manufacturer is sterile, no need to filter and sterilize. If it is found that there is a suspension in Gibco bovine serum, such as found in Gibco North American fetal bovine serum, the serum can be added to the culture solution and filtered together. Do not directly filter the serum.
3, bottled Gibco fetal bovine serum South American thawing need to use the thawing method: -20 ° C to -70 ° C low temperature refrigerator serum in a 4 ° C refrigerator dissolved for 1 day. Then moved to room temperature, after all dissolved and then repackaged, generally 40 to 45 ml can be dispensed in a 50 ml sterile centrifuge tube. During the dissolution process, it should be shaken regularly (be careful not to cause bubbles), so that the temperature and composition are uniform, and the precipitation is reduced. Do not directly thaw the serum from -20 ° C into 37 ° C, so that because the temperature changes too much, it is easy to cause protein aggregation and precipitation.
4. Heat inactivation refers to the serum that has been completely thawed at 56 ° C for 30 minutes. It should be shaken regularly during heating. The purpose of this heat treatment is to inactivate the complement components in the serum. Unless necessary, this heat treatment is generally not recommended. Because heat treatment will cause a significant increase in serum sediments, and will also affect the quality of serum. Complement participates in the reaction: cytotoxicity, smooth muscle cell contraction, mastamine and platelet release of histamine, enhance phagocytosis, promote lymphocytes and macrophages Cells undergo chemotactic and activation.
5. Precipitates and flocs in serum: mainly caused by denaturation of serum lipoproteins and fibrin in serum after thawing. These flocs do not affect the quality of the serum itself. It can be removed by centrifugation at 3000 rpm for 5 minutes. Can be processed. "Little black spots" under the microscope: After heat-treated serum, the formation of precipitates is significantly increased. Some sediments are observed under the microscope like "small black spots" and often mistaken for serum contamination. In general, this small black spot does not affect cell growth, but if serum quality is suspected, stop using it immediately and replace another batch of serum.
Summary of common problems encountered in the use of Gibco fetal bovine serum
First, serum inactivation problem
1. Q: Must the serum added to the medium be inactivated?
A: It is not necessary. I will see what experiments I have done. When I use Gibco South American serum 10270106, it is not inactivated.
2. Q: Is Gibco fetal bovine serum New Zealand (Gibco New Zealand serum 10091148) inactivated at 56 ° C for 30 minutes?
A: If used to culture most tumor cells, serum is generally not required to be inactivated. For example, Gibco New Zealand fetal calf serum is often used without heat inactivation, which can effectively preserve growth factors in serum. However, if used to culture cells with surface complement receptors, such as endothelial cells, serum is inactivated, typically 56 ° C for 30 min.
3. Q: I want to grow trophoblast cells. Is Gibco North American fetal bovine serum inactivated?
A: Gibco fetal bovine serum used in general cell culture, such as Gibco ES-specific fetal bovine serum, is not inactivated. If it is necessary to inactivate, you can directly purchase the Australian hot version of Gibco fetal calf serum, and the heat inactivated by Gibco Australian fetal calf serum is generally inactivated. The price of inactivated Gibco serum is generally much higher than the price of non-inactivated Gibco serum.
4. Q: I use the virus supernatant to transfect the cells. Does the serum used for culture need to be inactivated? Because there may be some complement and antibodies in the serum, will it affect transfection? I think that the inactivated serum contains some antibody complement that may be combined with the virus, affecting transfection, is it correct? The cells are monocytic leukemia cells, and the virus is the viral supernatant collected by the viral packaging cell PT67. Why use serum-free medium plus virus for several hours, what is the purpose?
A: The serum must be inactivated. It can be incubated with serum-free medium plus virus for a few hours before adding serum. Avoid interference of the protein with the virus and host binding process, usually 2-6 hours, depending on the state of the cell. Serum does not necessarily need to be inactivated, the purpose of inactivation is to inactivate the complement in serum! This depends on the actual situation. If you are not sure, then it will be inactivated, and it will not be troubled for 56 degrees and half an hour.
5, Q: (1) I bought Gibco fetal bovine serum North America, do you want to inactivate it? Some sayings are needed, some say that they can be added to the medium after thawing directly; I am preparing trypsin solution, using D-PBS, is it related?
A: The hot inactivation of serum is a topic of controversy that many people are interested in. Most laboratories perform heat inactivation of serum as a routine because there are two roles, the first being inactivation of complement and the second being inactivation of the mycoplasma that may be present in the serum. However, the experimenter did not consider the negative effects of heat treatment on growth factors, amino acids and other components in serum.
When I was working on Gibco fetal bovine serum in New Zealand, there was a water bath failure during inactivation. The temperature rose to 80 ° C and the serum became gelatinous. I reprocessed another Gibco fetal calf. Serum South America, comparing the two sera, found that high temperature directly affects the antibody protein contained in the serum. Inactivation at 56 ° C for more than half an hour will certainly damage the protein inside. Next time I have the opportunity to try to extend the inactivation time to see how big the impact is. After the heat is inactivated, the serum is placed in the four-degree refrigerator for a long time, and precipitation will occur, which is often considered to be microbial contamination or black-gel parasite contamination. In order to verify whether there is any pollution, the serum is often incubated at 37 ° C, but the protein in the serum will be further precipitated. Finally, microscopic examination, aseptic culture test, and Gram stain test are very troublesome.
I have read a document that mentions that 70% of experimenters believe that inactivation is a matter of course. The recommended temperatures for routine inactivation range from 45 ° C to 62 ° C, while the time ranges from 15 minutes to 60 minutes. The most common method used is heat treatment at 56 ° C for 30 minutes. With the improvement of serum collection, processing and processing technology, many of the reasons that were previously considered to be heat inactivation are no longer valid. Only a few researchers who performed heat inactivation on serum confirmed the effectiveness and necessity of this step in the experiment. Sex. Someone compared 11 different cell lines and found that heat inactivation negatively affected the growth of six cell lines (HBAE, MDBK, Vero, fibroblast, MRC-5), three cell lines (FOX-NY, MDCK and CHO-K1) were not affected by heat inactivation, while only two cell lines (Balb/3T3, Sp2/0Ag14 hybrid) showed a slight improvement in cell growth after heat inactivation. Therefore, under normal operation, heat inactivation usually does not significantly promote cell growth.
You can find out, Gibco North American serum 16000044 is taken out from the -20 °C refrigerator and thawed at room temperature, then mixed into two parts, one inactivated, one inactivated, compare the two Gibco North American serum to the cells Whether it has an impact. Then determine if it is inactivated or not. This process can take up to a week at most. At the same time, you should also consider whether serum inactivation has any effect on your subsequent experiments.
Q: (2) Gibco Australia serum 10099141 serum after dispensing is taken out from -20 °C and allowed to liquefy at 4 °C, but Gibco Australia serum 10099141 serum is turbid and precipitated. Is this normal?
A: Normal.
Second, the preservation of Gibco fetal bovine serum
1. Q: Can the fetal calf serum dedicated to GIBCO embryonic stem cells (Gibco ES-specific serum 16141079) expired in June 2016 be used in May 2017?
A: Only try it. If you have been frozen at -20 °C, you should still be able to use it. The cell line should be no problem, the original can't.
2. Q: Will I naturally dissolve the Gibco Australian serum 10099141 and 1640 medium, which I can't use today, I will use it tomorrow, I don't want to put it in the refrigerator, let it go at room temperature next night? How much medium is added to the 100 ml culture flask?
A: You can put 4 °C. 4-5ml.
3. Q: Can I continue to use Gibco South American serum for 3 months in a 4°C refrigerator? The cost of related cells and other reagents is relatively high and it is not easy to try.
A: Gibco fetal calf serum that needs to be preserved for a long time must be stored in a low temperature refrigerator at -20 to -70 ° C. Do not store in a refrigerator at 4 ° C for more than 1 month.
Third, the temperature problem of Gibco bovine serum inactivation
1. Q: Because the laboratory water bath is out of control, when the serum is inactivated, the temperature reaches 61.7 ° C. Can this serum be used?
A: How long? It should be OK in a short time. I have heated it for more than an hour and still use it.
2. Q: When I inactivate Gibco fetal bovine serum, the induction cooker is set at 70 ° C. In this case, the temperature is adjusted to 56 ° C and the serum is released. After forgetting, the serum is put in, so inactivated. The temperature must have exceeded 56 ° C. I wonder if this has any effect on serum?
A: The recommended temperature for routine inactivation is between 45 ° C and 62 ° C, and the time varies from 15 minutes to 60 minutes. The most common method is heat treatment at 56 ° C for 30 minutes. Look at what your cells are. In general, it is estimated that the problem is not big. If the precious cells are precious, be careful. The purpose of heat inactivation is to remove heat-sensitive substances such as complement in serum. In addition to complement inactivation, heat treatment also has an inactivation effect on mycoplasma that may be present in serum. It seems that heat inactivation is not advocated now. Heat inactivation often has a negative impact on serum products. Heating of serum often leads to precipitation in serum. In addition, studies have confirmed that heat inactivation steps weaken fetal bovine serum and calf The adhesion of serum to cells.
4. Can FBS replace human AB serum?
Q: I am currently working on the culture of human peripheral blood b lymphocytes. The literature requires the use of human type AB serum, but I don't think many agents have it. I want to replace FBS, but I don't know if it is possible. I am worried about immune rejection first, but after I have checked some information, it should not (I think). But I can't TRY again. Because the cytokines are really too expensive, so consult. Thank you!
A: You'd better do it according to the literature. Unless your series of experiments prove that the results of your substitutes are the same as in the literature (you still need to use the reagents in the literature). There are many factors influencing cell culture, especially the composition of the medium.
Five, serum preparation methods
1. Q: My experiment needs to prepare some serum for culturing cells. Does anyone know what preparation process is needed for culturing serum?
A: I prepared serum to be careful. We generally do not prepare fetal bovine serum by ourselves. We purchased Gibco bovine serum, Gibco New Zealand serum 10091148, which is very effective and stable. If the aseptic conditions are good, use a static precipitation method.
2, Q: In general, we use Gibco fetal bovine serum before use, I want to use the blood of the rat, do not know whether to inactivate?
A: You directly take the collected blood in a centrifuge tube at 4 ° C overnight, centrifuge, take the supernatant, in sterile filtration, can also be inactivated, and then frozen and stored, when used.
3. Q: If it is inactivated, will it damage some factors in serum?
A: 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. Heat inactivated serum is recommended for immunological studies, culture of ES cells, insect cells, and smooth muscle cells. Inactivation is generally not damaging to some of the factors in the serum.
6. The use of Gibco bovine serum in primary culture of cardiomyocytes
1. Q: In the primary culture of cardiomyocytes, it is necessary to stop the digestion of trypsin with serum-containing medium. I am using a culture solution containing 10% serum, but I have recently seen a cell culture courseware for North Medical College. I found that 1% serum culture solution was used to stop digestion. I would like to ask if 1% is acceptable. Is the effect guaranteed? This really saves a lot of serum.
A: About the FBS problem of cardiomyocyte culture:
(1) 1% serum complete medium can not, depending on the amount of trypsin. However, it is not possible to culture the cardiomyocytes in the Yuan Dynasty. 10% of FBS complete medium is not very effective. It requires high concentration of FBS, about 20%, to start cardiomyocyte culture. However, there is another problem. Fibroblasts are superior in FBS complete medium. For cells, it is necessary to add an appropriate amount of BrdU to the culture medium to inhibit growth and to purify the cardiomyocytes.
(2) The role of FBS is not only to neutralize trypsin, but some proteins in FBS, such as α-macroglobulin, inhibit pancreatic enzyme activity.
(3) The use of FBS in the culture of cardiomyocytes in the Yuan Dynasty has been paid attention to: the initial use of high-concentration FBS of about 20%, and then gradually reduced to serum-free DMEM/F-12 medium.
2. Q: The amount of trypsin used is 0.08%, and 0.08% of collagenase is mixed. Is it necessary to say that the high concentration of FBS is decreasing in cell culture? Do you need such a high concentration to terminate in digestion? Also, my BRDU is used in 48 hours, because it is worried that the damage is too great. How long can it last?
A: I stopped with 10% FBS, then the differential rate was 1h, and then I still used 10% FBS HG-dmem. Without brdu, the cardiomyocytes are still more stable and stable. I can use it on the 3rd day. If you look at it in the evening, you will have a beat.
Seven, serum and medium types and brand issues
1. Q: Which company's products are better for cell culture of fetal bovine serum? Some people use PAA or Hyclone, are these two different from Gibco or sigma?
A: If you are growing very fast cells such as pa317, 293, c6 and other malignant tumor cells, you can usually get domestic serum. If it is a primary cultured cell, it is best to use fetal bovine serum or standard fetal bovine serum, Hyclone. The quality of the company's serum is not as good as GIBCO (same serum). The domestically produced Hangzhou Sijiqing and Gansu Minhai (China-US and capital) are good. Some cells (such as sf9, 293 and some other meta-cells), much better with Gibco than the other two, will greatly speed up the test. For some other cells (such as: vero, MDCK, pk), all seasons, Hyclone's calf serum is enough! In addition, Gibco's depends on the origin, Gibco serum prices vary from place to place. Among all these serum brands, Gibco serum prices are higher and the quality is relatively stable.
2, Q: Recently, I have to order a bottle of Gibco North American serum 16000044. I used calf serum before the laboratory and I didn't buy Gibco fetal bovine serum. Which company is better? I have asked about reagent companies, there are two kinds: one is PAA (classic and superior), and the other is Hyclone (only common grade, and is New Zealand). Reagent companies recommend the use of PAA superior grade, but many documents use Hyclone, the company said that because Hyclone is not imported from the United States. Which kind of fetal bovine serum is better?
A: The effect of the Minhai is not bad. If you don't worry about domestic production, then buy imported. There are many companies that import, and Gibco New Zealand's serum works well. Both Hyclone and Gibco are fine. The people's sea seems to be better than the four seasons. The feeling of recovery is also good.
3. Q: What is the difference between Sijiqing “no phage, low endotoxin fetal bovine serum†and “no mycoplasma fetal bovine serum� Which one is better for culturing tumor passage cells?
A: You can use the mycoplasma-free Gibco Australian fetal bovine serum.
4. Q: Which type of 1640 medium and fetal bovine serum are used for SKBR-3 cell culture?
A: I have been using GIBCO's 1640. You can buy a 1*10L package containing HEPES. The fetal bovine serum is also used in GIBCO, 10%.
Eight, Gibco cattle serum contamination of phage
1. Q: Does anyone know how to avoid phage contamination during the manufacturing process of calf serum, and how can the bovine serum of contaminated phage be able to remove phage?
2. Q: I also want to know that most of the serum I use has phage. How does it affect cell culture?
A: I don't know what serum you are using. We use Gibco embryonic stem cell-specific serum 16141079, without any phage. It is recommended that you immediately change the fetal bovine serum. It is recommended that you use Gibco fetal calf serum. The specific product number is: Gibco South American fetal bovine serum (Gibco South American serum 10270106), Gibco North American fetal bovine serum (Gibco North American serum 16000044), Gibco Australian fetal calf Serum (Gibco Australia Serum 10099141), Gibco New Zealand Fetal Bovine Serum (Gibco New Zealand Serum 10091148), Gibco ES-specific fetal bovine serum (Gibco ES-specific serum 16141079).
Nine, serum concentration of the problem
Q: When adding serum in 1640, add more 20ml Gibco New Zealand serum 10091148, about 18%, previously added 10ml, check the online recommended 10%-15%, is it related?
A: I think that in general, the large fluctuation of serum concentration has a great influence on the state of the cells. It is recommended to add 90ml1640 to 10%. The serum concentration is large, of course, the cell state feels better, but the high concentration of Gibco Australia serum 10099141 may change the gene expression profile of the cells, affecting the results of subsequent experiments. It is good to culture snuff cancer cells at a concentration of 10%.
X. Q: Do you add serum when adding MTT? Is serum-free medium used when dosing?
A: My medicine is diluted with a medium containing Gibco Australian fetal bovine serum. The serum-free medium is toxic or inhibiting to the cells, and a serum-free model is created in the cells. Originally there is a serum supply, if the drug can not fight, then the drug has no clinical value, this is my understanding.
XI, serum problems in PC12 cell culture
Q: I am preparing to buy undifferentiated PC12 cells from Shanghai Cell. I need inactivated horse serum. It is very difficult to buy serum now. It seems that the customs has a blockade. The agent said that I originally purchased Gibco's horse. Surum, can't be bought now, so it's easy to find a Hyclone company that can currently enter the serum. Is there a kind of Donor Equine serum?
A: Hyclone's Donor Equine serum can be used. I used this one. I bought it at Dingguo (Chongqing Office), 100ml is about 140 yuan, I don't remember. It was at the time it was cheaper than other imported horse serum. In addition: I bought it and inactivated it.
Twelve, the preparation of AB serum
Q: I want to isolate AB serum from human peripheral blood for the culture of B lymphocytes. Thank you for your advice. Human blood is hard to come by.
A: Is it the serum of AB blood type people? This serum has no anti-type A antigen antibody and no anti-type B antigen antibody. When the serum is separated, the collected blood is injected into a test tube, and after the blood is coagulated, the serum is centrifuged. The collected blood can be placed in a 37 ° C water bath to promote blood coagulation and reduce the setting time. The centrifugation can be performed at 2500 to 3000 rpm for 10 minutes to separate the serum. After separation, the serum is aspirated and stored. The amount of serum separated can be calculated as about 50% of the collected blood, because red blood cells account for about 50% of the total blood. Of course, the entire process should pay attention to aseptic operation.
13. Does Gibico's fetal bovine serum contain sugar?
Q: I want to do a test for sugar-induced apoptosis. 20% fetal bovine serum was added to the cell culture medium. Therefore, whether or not the fetal bovine serum contains sugar has a great influence on the final concentration of the sugar in the culture solution of my experiment. Call today to ask GIBCO's technical consultant to answer that my sugar concentration is less than 5μg/ml, so it is basically considered to be sugar-free. But today I sent fetal bovine serum to our hospital for sputum examination, but the result was: 6.2mmmol/l (glucose oxidase method). Is it a method of detecting the blood glucose concentration of human serum that cannot be used to check bovine serum? (Another tea incident)? The laboratory did not give me an answer.
A: It should be sugar-free. Please refer to the gibco website: http:// I made a mark on the fifth page. Just click on the link to see its manual page. I think we must take the company's instructions as the standard. As for why the laboratory has errors in the measurement, I think it may be that the samples are different and need to be corrected with the mark. Specifically, the comrades in the laboratory need to explain.
14. The serum or medium produces color or sedimentation problems.
1. Q: I used Gibco fetal bovine serum. After a 30-minute inactivation at 56 °C, a small amount of flocculent white precipitate appeared. Is it pollution? Can you still use it? The date of production of the serum is July 2006 (now June 11, 2007).
A: It should be a small problem. You can take a small amount of serum into a petri dish and incubate at 37 ° C overnight to see if it is contaminated. There are many reasons for the presence of precipitates in serum, but the most common cause is due to the degeneration of lipoproteins in serum, and fibrin (one of the proteins that form blood coagulation) is also present in serum after blood thawing. It is also one of the main causes of sedimentation. However, these flocculent precipitates do not affect the quality of the serum itself. If you want to remove these flocculent deposits, the serum can be dispensed into a sterile centrifuge tube, centrifuged slightly at 400g, and the supernatant can be added to the medium for filtration.
2. Q: I measured the proliferation of the cells by MTT method, and lysed the cells with DMSO. It was found that adding DMSO to the wells formed milky white (using 1640 medium containing fetal bovine serum, because there is no centrifuge from the plate, so There was no DMSO added directly to the medium). This was not previously seen with media containing calf serum. How to solve it?
A: Why do you need to centrifuge with a centrifuge? Are you raising suspended cells? If it is adherent cells, dump the MTT directly, then add DMSO, we just do it, the effect is very good! And if you measure the proliferation of cells, if you do not remove MTT, add DMSO, the error should be great! Sometimes it's not necessarily the cause of serum, it may be related to the time of your MTT placement or other ingredients!
3, Q: (1) GIBCO fetal bovine serum 500ml, today unfractionated without direct mixing, the result is that the first few tubes are clear, followed by brown, I wonder if there is any impact? What is the impact? Is the composition of the front good or the brown ingredient good?
A: There must be. It is best to remix and repack, I think the active ingredients will be concentrated in the brown color section.
Q: (2) Why is brown?
A: The more brown in the serum may be due to the higher content of red protein.
Q: (3) Can serum be stored after inactivation? I was inactivated the day before yesterday, but it was not added to the medium, but was placed in the refrigerator. Can it be used directly next time? Still inactivated again?
A: Of course, if there is more, it is best to put it at -20 °C after the package, and then thaw it again when it is used again, and it is not necessary to inactivate it. It is best to take a tube with a tube and a little serum can be placed at 4 °C. When disassembling, be careful not to overfill it to avoid spillage.
Fifteen, pollution and filtration problems
1. Q: I suspect that the Gibco embryonic stem cell-specific fetal bovine serum (Gibco ES special fetal bovine serum) is contaminated. I want to filter it with a filter. Can I filter it with a 0.22μm filter? How much influence on serum properties? Have you done it?
A: It can be filtered. The serum is 0.22μm or 0.1μm filtered and sterilized when it leaves the factory. If you want to prove that there is no need to filter the bacteria, it is so troublesome to put it at 37 °C overnight. If it is contaminated by microbial contamination, it will become turbid. If it is still clear, it means no problem. Serum in the laboratory is difficult to filter directly, usually added to the medium and filtered. When we prepare the culture medium in our laboratory, we use a membrane to filter. Generally, if 1-2 bottles of medium are prepared, a filter and a 30 ml syringe can filter 50 ml of calf serum. Serum can also be added to the medium if the medium is prepared in large quantities, and filtered together using a 0.22 micron large filter membrane.
2. Q: I suspect that the completely 1640 culture solution I used is contaminated. I am going to re-filter and disinfect. Is it necessary to supplement the fetal bovine serum after filtration? How do you add it?
A: The completely 1640 culture solution is contaminated. If it is mycoplasma, the filtration has no effect, and the mycoplasma can pass through the filter. We use 1640 liquid, which is 500ml after dosing, and then other packs of 100-200ml, the current use is because the serum is more expensive than 1640, if you want to filter, it is recommended that you add the original proportional serum, because Gibco bovine serum It won't pass the filter easily. The main thing is to find the cause of possible pollution.
3. Q: Is the medium supplemented with Gibco bovine serum double-antibody still contaminated and filtered?
A: It is recommended not to use it. If it is contaminated with the addition of a double antibody, the possibility of bacterial contamination will be less. General filtration cannot remove the contamination of viruses or some fungi.
4. Q: I am going to re-filter the completely used 1640 culture solution. I don't know if the serum component in the culture solution can pass through the filter membrane. Is it still necessary to supplement the fetal bovine serum after filtration? How do you add it?
A: It can pass, but it will be very slow as the amount of liquid increases. If it is not pressurized, it will be more troublesome. It is better to use low protein adsorption, otherwise the loss is relatively large.
XVI. Q: Can I add Gibco fetal bovine serum when suspension cells are cultured? What happens if I add it? Why can't I add Gibco serum when I am in suspension?
A: Serum is one of the important culture components in cell culture media and has a wide range of effects on cell growth. When cells are cultured, we usually add 10% serum. The quality of serum is critical to the success of cell culture. In general, bovine serum includes the following components: growth factors (such as hormones, interleukins, etc.), they can promote cell growth; attaching factors, they can promote cell adhesion, often only cell adherence can increase value (suspension culture Except for cells; proteins (such as serum albumin, globulin, etc.) can be used as both nutrients and pancreatic enzymes; there are many substances with unknown components. Serum can also be detoxifying, such as the toxicity of fatty acids, heavy metals and certain proteases. Serum can also be cells from mechanical damage. Therefore, serum is essential whether it is adherent or suspension cells. Unless serum is cultured as required by the experiment, for example, to synchronize the cells, we will use serum-free culture. Simply saying that suspension cell culture can not add Gibco fetal bovine serum does not make much sense.
17. The amount of serum needed to neutralize the digestive juice
Q: After digesting cells with trypsin, serum neutralization is required. What is the ratio between trypsin and serum required for this specific neutralization?
A: I have been doing experiments for a long time. I really didn't think about the corresponding relationship between trypsin and serum. But think about it, this should have no fixed ratio. The variety and origin of serum are different, and the ingredients must be different. How can we determine the ratio of the ratio to pancreatic enzyme? When we digest the cells, if it is passaged, after the cells are deformed, aspirate the trypsin and add the appropriate amount of hanks or whole culture. This amount depends on your preference and the convenience of blowing cells. Generally, the digestion can be stopped. There will be no further things to continue to digest. If it is from the tissue digested cells for primary culture, I generally use the whole culture to stop, and add a lot, 0.25% trypsin, with 10% serum, the ratio of 1:2 should be sufficient. Of course, the total culture is 2, generally when I raise cells, I will use the cheaper serum made in China to prepare the whole culture, which is used to stop digestion. There are several advantages: First, the effect of stopping digestion should be better than hanks liquid. It is also beneficial to maintain cell viability. And this part of the liquid will be removed by centrifugation, the serum is cheap, and it will not be distressed when it is centrifuged. Use serum when raising cells. Personal point of view, only for reference.
Eighteen, Gibco fetal bovine serum suspension problem in New Zealand
1. Q: It is found that there are many small black spots in the background of the cultured cell bottle, and there are some floating objects in the culture solution. I looked at the previous post and thought it was a collagen worm. I intend to change the liquid. Before changing the liquid, I carefully looked at the culture solution of the previous day. The naked eye found that there were suspended particulate matter in the culture solution (not much), so I observed it under the microscope and there was some suspension in the fresh culture solution. Small particles, not much. (The culture medium is R1640 + 20% Gibco calf serum + 1% double antibody). Then the calf serum stored in 4 ° C is taken out and observed, and 5 or 6 white small spherical substances are visible to the naked eye. The bottle is suspended slightly below. Under the microscope, there are also a small amount of things that don't know anything. The calf serum is Hyclone New Zealand and the label has been filtered through a 2μm filter. According to the above culture solution, does it mean that the culture solution has been contaminated? If it is normal serum is not under the microscope should not see debris, even a very small amount? According to the description of the above serum, is it that the serum also has problems, can it be used? Supplement: The growth state is not good since cell culture. When buying serum, the manufacturer stated that it does not need to be inactivated, so it is not inactivated.
Answer: (1) Gibco serum should be stored at -20 °C. When thawing serum, please follow the step-by-step thawing method (-20 °C to 4 °C to room temperature). If the temperature is too large when the serum is thawed, the experiment shows that it is very easy to produce sediment. .
(2) There are many reasons for the appearance of precipitates in Gibco Australia serum 10099141, but the most common cause is due to the degeneration of serum lipoproteins, which are also present in serum after thawing of Gib fetal fetal bovine serum. In the case of Gibco New Zealand Fetal Bovine Serum 10091148, a precipitate may also be formed.
(3) If you can't use one bottle at a time, it is recommended that you fill the serum aseptically and put it back into the frozen. If stored at 4 °C, do not exceed one month, store at 2-8 °C, the serum in each Proteins and lipoproteins that may aggregate to form a precipitate or visible turbidity.
(4) When thawing Gibco fetal bovine serum, please shake it at any time to make the temperature and composition uniform, reducing the occurrence of sedimentation.
(5) The reason for the exclusion of Gibco serum (eg Gibco New Zealand fetal bovine serum 10091148) is to consider the problems of laboratory supplies and aseptic processing.
(6) Bacterial viruses, chlamydia, and black worms are also common. If the cells die too much, it is recommended to change the culture solution.
2. Q: Today, when the culture solution was mixed, it was found that there were small debris-like floating objects in the Gib serum, and the serum was still clear and not turbid. I don't know what it is. I asked the seniors of the laboratory and said that they can still be used, but I still don't worry, I don't use serum. Helping everyone in the garden, this may be a problem with the serum? My serum used for less than a month, all seasons.
A: Possible reasons: (1) Votex is not enough when the culture solution is configured. It is clear at the time, but it will be precipitated after a while; (2) fungal contamination.
Nineteen, the problem of a large number of cell death after replacing serum-free medium
Q: I used Lipofectamine 2000 to transfect osteoblasts and replaced them with serum-free medium before transfection. The original conditional model is good and the transfection efficiency is acceptable. But when the winter holiday came back, something strange happened: the cells grew well in the serum-containing medium, and died when the serum-free medium was changed. You can see more cells floating in about 4 hours. But I changed the serum-free medium, even if I put it there for 10 hours, it would be no problem. Already changed the medium of different batches, even the straw has been changed, but it is not, what is going on?
Answer: (1) After two weeks, the culture solution should be supplemented with glutamine or re-dosed. The transfection should be free of antibiotics.
(2) Confirm the operation method and environment, it is recommended to check it.
(3) Lipofectamine2000, liposome is very toxic, if there is a plasmid involved in cell damage, if Lipofectamine2000 is placed at room temperature, the cells are larger, and the holiday refrigerator is over-powered.
(4) Temperature, c02 concentration, and humidity of the incubator.
Twenty, the definition of complete culture fluid
Question: "Is the term "completely cultured fluid" refers to the culture medium with serum added? I am doing experiments with drug-containing serum, which involves the problem of serum addition. So I want to understand whether the "complete culture solution" referred to in the article is a drug-containing serum.
A: The stock solution refers to filtration and sterilization after configuration. Incomplete culture fluid refers to a stock solution that does not contain serum, such as Gibco North American fetal bovine serum, and other ingredients have been supplemented. A complete culture solution refers to an incomplete culture solution added to serum (for example, Gibco fetal bovine serum North America) and is referred to as a complete culture solution.
Twenty-one, a summary of serum related knowledge.
Precautions for using Gibco fetal calf serum:
Serum is one of the most important elements in cell culture. The following are the problems that are often encountered in the laboratory using Gibco fetal bovine serum, such as Gibco North American fetal bovine serum, for your reference only:
1. The best way to preserve Gibco bovine serum:
It is recommended that Gibco serum should be stored at -5 ° C to -2 ° ° C. However, if stored at 4 °C, do not exceed one month.è‹¥æ‚¨ä¸€æ¬¡æ— æ³•ç”¨å®Œä¸€ç“¶ï¼Œå»ºè®®æ‚¨æ— èŒåˆ†è£…血清至æ°å½“çš„çèŒå®¹å™¨å†…，å†æ”¾å›žå†·å†»ã€‚
2ã€å¦‚何解冻Gibco胎牛血清æ‰ä¸ä¼šä½¿äº§å“è´¨é‡å—æŸï¼š
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4ã€ä½•è°“çƒç活：
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5ã€å…³äºŽGibco胎牛血清的ç活：
有必è¦åšçƒçæ´»å—？
实验显示，ç»è¿‡æ£ç¡®å¤„ç†çš„çƒçæ´»Gibco血清，对大多数的细胞而言是ä¸éœ€è¦çš„。ç»æ¤å¤„ç†è¿‡çš„Gibco澳洲胎牛血清10099141（Gibco澳洲血清）对细胞的生长åªæœ‰å¾®å°çš„ä¿ƒè¿›ï¼Œæˆ–å®Œå…¨æ²¡æœ‰ä»»ä½•ä½œç”¨ï¼Œç”šè‡³é€šå¸¸å› ä¸ºé«˜æ¸©å¤„ç†å½±å“了血清的质é‡ï¼Œè€Œé€ æˆç»†èƒžç”Ÿé•¿é€ŸçŽ‡çš„é™ä½Žã€‚而ç»è¿‡çƒå¤„ç†çš„血清，沉淀物的形æˆä¼šæ˜¾è‘—的增多，这些沉淀物在倒立显微镜下观察，åƒæ˜¯â€œå°é»‘点â€ï¼Œå¸¸å¸¸ä¼šè®©ç ”究者误以为是血清éå—污染，而把血清放在37℃ 环境ä¸ï¼Œåˆä¼šä½¿æ¤æ²‰æ·€ç‰©æ›´å¢žå¤šï¼Œä½¿ç ”究者误认为是微生物的分裂扩增。
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6ã€è¡€æ¸…相关知识：
如何é¿å…Gibco胎牛血清沉淀物的产生？
我们建议您在使用Gibco牛血清，例如Gibcoå—美胎牛血清的时候，注æ„下列的æ“作:
(1)解冻Gibco胎牛血清å—美时，请按照所建议的é€æ¥è§£å†»æ³•(-2O℃至4℃至室温)，若血清解冻时改å˜çš„温度太大(如-20℃至37℃)，实验显示éžå¸¸å®¹æ˜“产生沉淀物。
(2)解冻Gibco胎牛血清时，请éšæ—¶å°†ä¹‹æ‘‡æ™ƒå‡åŒ€ï¼Œä½¿æ¸©åº¦åŠæˆåˆ†å‡ä¸€ï¼Œå‡å°‘沉淀的å‘生。
(3)请勿将Gibco北美血清16000044置于37℃太久。若在37℃放置太久，血清会å˜å¾—混浊，åŒæ—¶è¡€æ¸…ä¸è®¸å¤šè¾ƒä¸ç¨³å®šçš„æˆåˆ†ä¹Ÿä¼šå› æ¤å—到æŸå®³ï¼Œè€Œå½±å“血清的质é‡ã€‚
(4)Gibcoå—美血清10270106çš„çƒçæ´»éžå¸¸å®¹æ˜“é€ æˆæ²‰æ·€ç‰©çš„增多，若éžå¿…è¦ï¼Œå¯ä»¥æ— é¡»åšæ¤æ¥éª¤ã€‚
(5)若必须åšGibco胎牛血清的çƒç活，å¯ä»¥ç›´æŽ¥è´ä¹°Gibco胎牛血清的çƒç活产å“，但是ç活的Gibcoè¡€æ¸…ä»·æ ¼è¾ƒé«˜ã€‚è‡ªå·±åˆ¶å¤‡Gibco胎牛血清澳洲çƒç活的时候，请éµå®ˆ56℃，30 分钟的原则，并且éšæ—¶æ‘‡æ™ƒå‡åŒ€ã€‚温度过高，时间过久或摇晃ä¸å‡åŒ€ï¼Œéƒ½ä¼šé€ æˆæ²‰æ·€ç‰©çš„增多。
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