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Contents of this article
- 1. The difference between cell culture medium and bacterial culture medium
- 2. Is the preparation of tris-hcl buffer toxic?
- 3. What are the similarities and differences between PIPES and HEPES as buffers?
- 4.The difference between hepes buffer and PBS
The difference between cell culture media and bacterial culture media
There are many classic culture media, among which DMEM, RPMI 1640, MEM, and DMEM/F12 are the most widely used media. Others such as M199, IMDM, L15 medium, etc. are also used for the culture of certain cells.
The following are the specific characteristics and applications of some culture media:
1. MEM cell culture medium
Also known as Minimal Eagle Medium Essential Medium), modified from Eagle's basal medium (BME) in 1959, deleting lysine and biotin, and increasing the concentration of amino acids. It is suitable for the growth of a variety of cell monolayers and has autoclave-sterilizable varieties. It is the most basic and widely applicable medium. However, due to its limited nutritional content, it may not necessarily be the best or most economical medium for specific cell culture and expression.
2. BME cell culture medium
Basal Medium Eagle, designed by Eagle in 1955, BSS + 12 amino acids + glutamine + 8 vitamins. Simple and easy to add, it is suitable for various passage cell lines and special research. On this basis, improved cell culture media varieties include MEM, DMEM, IMDM, etc.
3. MDM cell culture medium
Guilber and Iscove modified Dulbecco's Medium into Iscove's Medium for culturing erythrocytes and macrophage precursors. This culture medium contains selenium, additional amino acids and vitamins, sodium pyruvate and HEPES. And potassium nitrate was used instead of ferric nitrate. IMDM can also promote the growth of mouse B lymphocytes, LPS-stimulated B cells, bone marrow hematopoietic cells, T cells and lymphoma cells. IMDM is a very nutrient-rich culture medium, so it can be used for rapid proliferation culture of high-density cells.
4. DMEM cell culture medium
DMEM is Eagle's medium modified by Dulbecco, originally designed for mouse fibroblasts. The amino acid concentration of DMEM is twice that of MEM, the vitamin concentration is 4 times that of MEM, and the double concentration of HCO3- and CO2 is used to provide a better buffering effect. The glucose content in the original formula was 1000 mg/L. Later, for the growth needs of certain cells, the glucose content was adjusted to 4500 mg/L. This is what everyone often calls low sugar and high sugar.
Low sugar is suitable for the culture of adherent-dependent cells, especially for the culture of tumor cells with fast growth and poor adhesion. High sugar is more suitable for high-density suspension cell culture. It is also suitable for clone culture with poor adhesion but does not want it to break away from the original growth point. It can also be used for the culture of myeloma cells in hybridomas and transformed cells transfected with DNA. For example, CHO cells express and produce hepatitis B vaccine, and CHO cells express EPO.
5. HamF10 cell culture medium
Designed by Ham in 1963, it contains trace elements and can be used when serum levels are low, and is suitable for clonal culture. F10 is suitable for hamster and human diploid cells, especially amniotic fluid cell culture.
6. DMEM/F12 cell culture medium
Ham's F12 is designed for cloning CHO cells at low serum concentrations and is now also widely used for colony formation rate Analysis and primary culture. F12 can also be mixed with equal volumes of DMEM to obtain a product that combines high concentration and diverse ingredients. This medium has been used in the cultivation of many primary cultures and more difficult to grow cell lines. Because it is rich in nutrients and can use less serum, it is often used as the basal medium for serum-free media.
7. RPMI-1640 cell culture medium
Developed by Moore and other scientists at Roswell Park Memorial Institute in 1967, it is designed for lymphocyte culture and contains BSS + 21 amino acids + 11 kinds of vitamins, etc. Now it is also used for the culture of suspension cells, such as mammals, special hematopoietic cells, normal or malignant leukocytes, hybridoma cells, and other lymphoblasts such as K-562, HL-60, Jurkat, Daudi, IM-9, etc. , T-cell lymphoma cells and HCT-15 epithelial cells can be used for reference.
8. McCoy5A medium
MeCoy was designed for sarcoma cells in 1959, with BSS+40 components. It can support the growth of a variety of primary grafts (such as bone marrow, skin, lungs, spleen, etc.). In addition to being suitable for general primary cell culture, it is mainly used for tissue biopsy culture, some lymphocyte culture, and some difficult-to-culture Cell growth support. For example, Jensen rat sarcoma fibroblasts, human lymphocytes, HT-29, BHL-100 and other epithelial cells.
9. M199 cell culture medium
In 1950, Morgan et al. designed a cell culture medium with a defined chemical composition, namely M-199. In addition to BSS, it contains 53 components and is a comprehensive culture medium, mainly used for culture of chicken embryo fibroblasts. This culture medium must be supplemented with serum to support long-term culture. M-199 can be used to culture cells from a variety of species and can culture transfected cells. Now it is widely used in virology and vaccine production.
10. L15 cell culture medium
L-15 culture medium is suitable for the culture of rapidly proliferating tumor cells and is used to culture tumor cell lines in the absence of CO2. . This culture medium uses a phosphate buffer system, the amino acid composition is further improved, and glucose is replaced by galactose.
Cell culture medium must contain sufficient nutrients to meet the materials and energy required for biochemical reactions such as new cell synthesis and cell metabolism. The main components of cell culture medium are water, amino acids, vitamins, carbohydrates, inorganic salts and other auxiliary nutrients. In addition, it may also contain serum, serum replacement ingredients, pH indicators, etc.
How to choose a culture medium, there are several suggestions for reference:
(1) The culture medium used to establish a certain cell line should be the preferred culture medium for cultivating such cells. You can check the references or consult when purchasing cell lines. You can also search on the websites of some biological companies. For example, the Invitrogen website has a Cell Line Database tool. Select the cell type you are interested in, and it will pop up recommended media, serum, transfection reagents, etc., and sometimes The optimized transfection procedure is very convenient.
(2) You may wish to try other media commonly used in laboratories. Many media can be suitable for a variety of cells.
(3) Select the culture medium according to the characteristics of the cell line and the needs of the experiment. For example, choose RPMI1640 for mouse cell lines.
(4) Use a variety of culture media to culture the target cells and observe their growth status. You can use growth curves, colony formation rates and other indicators to judge, and select the best culture medium based on the experimental results. This is the most objective method. , but it is more cumbersome.
Analysis of common problems during the use of cell culture media:
Since the suitable pH for most cells is 7.0~7.4, deviation from this range may have harmful effects on cell growth. However, the pH requirements of various cells are not exactly the same. Primary cultured cells generally have poor tolerance to pH changes, while unlimited cell lines have strong tolerance. Therefore, during primary culture, the buffer system in the culture medium is more important. Generally, cell culture media use a balanced salt system, but different culture media or the same series of culture media use different balanced salt systems. For example, the 199 series and the MEM series both have Hanks' system culture media and Earle's system culture. base. Some media are not the conventional balanced salt system mentioned above, such as RPMI1640 culture medium and F12 culture medium. The balanced salt system of MEM low serum medium is also not a conventional balanced salt system. The buffering capacity of this balanced salt system is stronger than that of a conventional balanced salt system.
There are many reasons for the decrease in pH value during cell culture. When cells grow very fast, the pH value usually drops quickly, which can be solved by timely passage, increasing the passage ratio, or reducing the amount of serum. In addition, the pH value usually drops quickly due to over-tightening of the culture bottle cap, insufficient buffering capacity of the NaHCO3 buffer system, incorrect salt concentration in the culture solution, bacterial, yeast or fungal contamination. At this time, the following methods can be used to solve the problem:
1) Increase the NaHCO3 concentration in the culture solution or reduce the CO2 concentration in the incubator. When the NaHCO3 content is between 2.0 g/L and 3.7 g/L, the corresponding CO2 concentration is 5~10%
2) Switch to a CO2-independent culture medium
3) Loosen appropriately bottle caps. Add HEPES buffer to the culture medium to make the final concentration 10~25 mM
4) Use a culture medium based on Earle's salt in a CO2 culture environment and culture it in an atmospheric environment For medium culture, use culture medium prepared with Hanks' salt
5) If it is caused by contamination, discard the culture or use antibiotics to sterilize it.
Is the preparation of tris-hcl buffer toxic?
When I extract lipid rafts from tumor cells, I use Modified HEPES buffer: 25mMHEPES-HCl, pH6.5, 150mMNaCl, 1mMEDTA, 1mMPMSF, protease inhibitor cocktail. But what I saw is Tris-HCl buffer. There is a difference between using them with the cell lysate. ? Or can it be replaced?
Tris-HCl buffer: The main components of Western and IP cell lysis buffer are 20mM Tris (pH7.5), 150mM NaCl, 1% Triton X-100, and sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, leupeptin and many other inhibitors. It can effectively inhibit protein degradation and maintain the original protein-protein interaction.
What are the similarities and differences between PIPES and HEPES as buffers?
Neither PIPES nor HEPES can form stable complexes with metal ions, so they are suitable for solution systems containing metal ions. But there are certain differences between them. In terms of solubility, PIPES is insoluble in water, while HEPES has good water solubility. In terms of buffer range, PIPES is acidic to neutral, and HEPES is neutral to alkaline. This is mainly because Due to the structural differences between the two, PIPES has two sulfonic acid groups, and HEPES contains a sulfonic acid group and a hydroxyl group. In addition, PIPES and HEPES have certain limitations in certain system applications. Therefore, when we select the above buffers, we need to comprehensively consider the suitability of the experimental system and the difference in properties between the two. We must learn to distinguish and understand the similarities and differences among these reagents, so that we can better promote the development and production of our different products. Desheng consistently adheres to this principle of not letting go of the slightest difference, so that it can continue to develop and produce better products.
The difference between hepes buffer and PBS
The pH buffer range of PIPES is 6.1-7.5. It is insoluble in water and soluble in NaOH aqueous solution. PIPES is different from buffers containing bis(2-hydroxyethyl)amino groups (such as Bis-tris, Bicine) in that it cannot form stable complexes with most metal ions and is suitable as a buffer in solution systems containing metal ions. Based on existing research results, PIPES can be used to purify tubulin using phosphocellulose chromatography, to purify recombinant GTP-binding proteins ARF1 and ARF2 by gel filtration, and as a buffer to crystallize transketolase from E. coli. In addition, because PIPES can form free radicals, it is not suitable for use in redox systems. In cation exchange chromatography, low-concentration PIPES buffers should be used because PIPES has a relatively large ionic strength and its pKa value is concentration-dependent.
HEPES
The pH buffer range of HEPES is 6.8-8.2. It is soluble in water and does not form stable complexes with metal ions. In most cases, it will not interfere with biochemical processes. HEPES is commonly used in various types of organisms. In the cell culture medium; in protein research, PIPES is often used as a component and eluent of the binding buffer in cation exchange chromatography; in DNA research, PIPES is used as a buffer for the system formed by calcium phosphate and DNA precipitate. AFM and buffers for electroporation experiments. In addition, HEPES has certain interference with the reaction between DNA and restriction enzymes, and is not suitable for use in Lowry's method to determine protein content. To sum up, both PIPES and HEPS are Good’s buffers and cannot form stable complexes with metal ions, so they are suitable for solution systems containing metal ions. But there are certain differences between them. In terms of solubility, PIPES is insoluble in water, while HEPES has good water solubility. In terms of buffer range, PIPES is acidic to neutral, and HEPES is neutral to alkaline. This is mainly because Due to the structural differences between the two, PIPES has two sulfonic acid groups, and HEPES contains a sulfonic acid group and a hydroxyl group. In addition, PIPES and HEPES have certain limitations in certain system applications. Therefore, when we select the above buffers, we need to comprehensively consider the suitability of the experimental system and the difference in properties between the two. In this way, you can better find the product you need when purchasing this Good’s buffer. Desheng will also give you an accurate positioning.
The above is all about the difference between tris and hepes, the difference between cell culture medium and bacterial culture medium, and the related content of trish. I hope it can help you.