1. Detection of cellular metabolic activity
The metabolic activity of the cells can also be measured to reflect the proliferation of the cells. The activity of lactate dehydrogenase increases during cell proliferation, and the active dehydrogenase can reduce the exogenous tetrazolium salt or Alamar blue to a color-reducing product. The absorbance of the dye-containing medium is read by a spectrophotometer or a microplate reader to measure the metabolic activity of the cells and detect the proliferation of the cells.
The four most common tetrazolium salts are: MTT, XTT, MTS and WST1, the reduction product of which is formazan.
(1) Tetramethylazozolium salt method (MTT): The commercial name of MTT is thiazole blue, which is a yellow dye. In 1983, Mosmann established the MTT colorimetric method for detecting cell survival and proliferation. The principle is that succinate dehydrogenase in living cell mitochondria can reduce exogenous MTT to water-insoluble blue-violet crystal, which is deposited in cells, and dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve the hyperthyroidism in cells, and its light absorption value can be measured by enzyme-linked immunosorbent assay, which can indirectly reflect the number of viable cells. The amount of MTT crystal formation is proportional to the number of cells in a certain number of cells. MTT can be used for all cell types, but MTT is insoluble in standard cell culture media and the formazan crystals it produces need to be dissolved in DMSC). Therefore, MTT is mainly used as an endpoint detection method. In addition, some studies have found that peroxide reduces the accuracy of MTT measurement, inhibits the reaction of MTT and O2- by nearly 95%, and the MTT lysate hyperthyroidism adsorbs on nanofibers, resulting in false negative results.
(2) Dimethoxazole yellow colorimetric method (XTT): XTT is a tetrazolium nitrogen derivative similar to MTT, which can be reduced by living cells to form a water-soluble orange-yellow formazan product, which does not form particles. The absorbance is directly detected by an enzyme-linked immunoassay analyzer, so it is faster, simpler and more sensitive than the MTT method.
However, the XTT aqueous solution is unstable and needs to be stored at a low temperature, and is now available. Since the metabolites of XTT are orange, some yellow metabolites and reagents in the culture system may affect their detection results. Like MTT, peroxide inhibits the reaction of XTT and O2- by nearly 95%, so it has a certain influence on the accuracy of XTT measurement.
(3) Inner Salt Method (MTS): MTS is a novel MTT analog. In the presence of the coupling agent PMS, MTs can be reduced to water-soluble colored formazan products by various dehydrogenases in living cell mitochondria. The color depth is highly correlated with the number of living cells within a certain range. Standard instrument detection. Its advantages are non-radioactive, fast, safe, convenient, flexible and specific, while overcoming the shortcomings of MTT and XTT.
(4) Tetrazolium monosodium salt method (WST-1): WST-1 is a water-soluble tetrazolium salt reagent, which is a compound similar to MTT. In the presence of an electron coupling reagent, it can be some in the mitochondria. Dehydrogenase reduction produces orange-yellow formazan. The more the cells proliferate, the darker the color; the greater the cytotoxicity, the lighter the color. WST-1 is an upgraded replacement for MTT and has significant advantages over MTT or other MTT-like products such as XTT, MTS, etc. First, the hyperthyroidism produced by MTT reduction by some dehydrogenases in the mitochondria is not water-soluble and needs to be dissolved by a specific solution: while the hyperthyroidism produced by WST-1 and XTT, MTS is water-soluble, Go to the subsequent dissolution step. Second, the hyperthyroidism produced by WST-1 is more soluble than the hyperthyroidism produced by XTT and MTS. Again, WST-1 is more stable than XTT and MTS. After adding WST-1 color, the plate can be read repeatedly with the microplate reader at different times, making the detection time more flexible and the experimental results more stable. In addition, compared with MTT and XTT, WST-1 has a wider linear range and higher sensitivity.
(5) Cell countjnè¬kit-8 (CCK-8): WST-8 is contained in the CCK-8 reagent. WST-8 is a newly developed water-soluble tetrazolium salt which is newer than WST-1. The detection principle is similar to WST-1, but it is more stable, more sensitive, more soluble and easier to preserve than WST-1. . The sensitivity of CCK-8 for cell proliferation and cytotoxicity assays is higher than that of MTT, XTT and MTS, especially for suspension cells and high-throughput drug screening. The CCK-8 method has low cytotoxicity and can be reused after cell detection. It has better practicability and can replace the MTT method and has a good application prospect.
(6) Alma Blue Method: Alma Blue detection reagent provides a simple, rapid, reliable and safe method for cell proliferation and cytotoxicity detection. Alamar blue exhibits purple-blue non-fluorescence in the oxidized state, and in the reduced state, it is converted into a pink or red fluorescent reduction product, which can be detected by a common spectrophotometer or a fluorometer, absorbance and fluorescence intensity and activity. The number of cells is proportional. Alamar blue is non-toxic and harmless to cells, does not affect cell metabolism, cytokine secretion, antibody synthesis, etc., and can continuously observe and further test the growth state of the same batch of cells.
MTT can be used for all cell types, but MTT is insoluble in standard cell culture media and the formazan crystals it produces need to be dissolved in DMSO. Therefore, MTT is mainly used as an endpoint detection method. The other three salts, like Alma Blue, are soluble and non-toxic. They can be used as a continuous monitoring tool to track dynamic changes in cell proliferation. Among them, XTT is less efficient and needs to add additional factors. WST-1 is more sensitive and effective, and can develop color faster than other salts. The sensitivity of Alamar Blue is also very high, as long as there are 100 cells in the well of the microplate. Tetrazolium salts and Alamar blue redox dyes are very convenient for use in a variety of instruments and high-throughput studies.
2. Detection of ATP content
ATP is a direct source of cellular energy. The ATP content in cells is strictly regulated. Dead cells or cells that are about to die are almost irresorbed by ATP. There is a strict relationship between ATP concentration and cell number measured in cell lysates or extracts. The linear relationship. Therefore, information on cell proliferation can also be obtained by detecting ATP.
ATP detection can be achieved by methods such as color reaction, fluorescence, chemiluminescence or isotope. The most widely used method is based on firefly luciferase (catalyzing luciferin oxidation, consuming ATP. If ATP is present, fluorescein will emit light, the luminescence efficiency is extremely high, and the luminescence amount has a good linear relationship with ATP content. Reflects the amount of ATP in the cells.
3. Live cell fluorescent labeling
Hydroxyfluorescein diacetate succinimide ester (CFSE) is a fluorescent dye that penetrates cell membranes. CFSE can easily penetrate cell membranes, accumulate in living cells and covalently bind to intracellular proteins. The CFSE releases a fluorescent substance, and these covalently bound fluorescent molecules rarely detach from the cells. CFSE-labeled cells can be used for in vivo observation for up to several weeks. When the cells divide, CFSE-labeled fluorescence can be evenly distributed into the two daughter cells, so the fluorescence intensity is half that of the parental cells. Thus, in a proliferating cell population, the fluorescence intensity of each successive cell is logarithmically decreasing, which can be analyzed by flow cytometry at 488 nm excitation and fluorescence detection channels.
4. Enhanced mark detection
Proliferating cells specifically express certain specific proteins, and specific monoclonal antibodies are used to recognize these proliferating cells. For example, in human cells, Ki-67, PCNA, etc. can serve as markers for cell proliferation. However, this method cannot perform high-throughput analysis due to the need for tissue slicing. However, this method is favored by cancer researchers because it can be used to detect the proliferation of tumor cells in vivo.
Beijing Baiou Bowei Biotechnology Co., Ltd. has set up a cell line, which is a center for cell lines. It is specialized in providing cell lines with low generation, short cycle and good activity. With a number of domestic and foreign research and development units, biomedicine, third-party testing institutions, research institutes have a good and stable long-term cooperative relationship! Welcome customers to inquire!
Http://
The metabolic activity of the cells can also be measured to reflect the proliferation of the cells. The activity of lactate dehydrogenase increases during cell proliferation, and the active dehydrogenase can reduce the exogenous tetrazolium salt or Alamar blue to a color-reducing product. The absorbance of the dye-containing medium is read by a spectrophotometer or a microplate reader to measure the metabolic activity of the cells and detect the proliferation of the cells.
The four most common tetrazolium salts are: MTT, XTT, MTS and WST1, the reduction product of which is formazan.
(1) Tetramethylazozolium salt method (MTT): The commercial name of MTT is thiazole blue, which is a yellow dye. In 1983, Mosmann established the MTT colorimetric method for detecting cell survival and proliferation. The principle is that succinate dehydrogenase in living cell mitochondria can reduce exogenous MTT to water-insoluble blue-violet crystal, which is deposited in cells, and dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve the hyperthyroidism in cells, and its light absorption value can be measured by enzyme-linked immunosorbent assay, which can indirectly reflect the number of viable cells. The amount of MTT crystal formation is proportional to the number of cells in a certain number of cells. MTT can be used for all cell types, but MTT is insoluble in standard cell culture media and the formazan crystals it produces need to be dissolved in DMSC). Therefore, MTT is mainly used as an endpoint detection method. In addition, some studies have found that peroxide reduces the accuracy of MTT measurement, inhibits the reaction of MTT and O2- by nearly 95%, and the MTT lysate hyperthyroidism adsorbs on nanofibers, resulting in false negative results.
(2) Dimethoxazole yellow colorimetric method (XTT): XTT is a tetrazolium nitrogen derivative similar to MTT, which can be reduced by living cells to form a water-soluble orange-yellow formazan product, which does not form particles. The absorbance is directly detected by an enzyme-linked immunoassay analyzer, so it is faster, simpler and more sensitive than the MTT method.
However, the XTT aqueous solution is unstable and needs to be stored at a low temperature, and is now available. Since the metabolites of XTT are orange, some yellow metabolites and reagents in the culture system may affect their detection results. Like MTT, peroxide inhibits the reaction of XTT and O2- by nearly 95%, so it has a certain influence on the accuracy of XTT measurement.
(3) Inner Salt Method (MTS): MTS is a novel MTT analog. In the presence of the coupling agent PMS, MTs can be reduced to water-soluble colored formazan products by various dehydrogenases in living cell mitochondria. The color depth is highly correlated with the number of living cells within a certain range. Standard instrument detection. Its advantages are non-radioactive, fast, safe, convenient, flexible and specific, while overcoming the shortcomings of MTT and XTT.
(4) Tetrazolium monosodium salt method (WST-1): WST-1 is a water-soluble tetrazolium salt reagent, which is a compound similar to MTT. In the presence of an electron coupling reagent, it can be some in the mitochondria. Dehydrogenase reduction produces orange-yellow formazan. The more the cells proliferate, the darker the color; the greater the cytotoxicity, the lighter the color. WST-1 is an upgraded replacement for MTT and has significant advantages over MTT or other MTT-like products such as XTT, MTS, etc. First, the hyperthyroidism produced by MTT reduction by some dehydrogenases in the mitochondria is not water-soluble and needs to be dissolved by a specific solution: while the hyperthyroidism produced by WST-1 and XTT, MTS is water-soluble, Go to the subsequent dissolution step. Second, the hyperthyroidism produced by WST-1 is more soluble than the hyperthyroidism produced by XTT and MTS. Again, WST-1 is more stable than XTT and MTS. After adding WST-1 color, the plate can be read repeatedly with the microplate reader at different times, making the detection time more flexible and the experimental results more stable. In addition, compared with MTT and XTT, WST-1 has a wider linear range and higher sensitivity.
(5) Cell countjnè¬kit-8 (CCK-8): WST-8 is contained in the CCK-8 reagent. WST-8 is a newly developed water-soluble tetrazolium salt which is newer than WST-1. The detection principle is similar to WST-1, but it is more stable, more sensitive, more soluble and easier to preserve than WST-1. . The sensitivity of CCK-8 for cell proliferation and cytotoxicity assays is higher than that of MTT, XTT and MTS, especially for suspension cells and high-throughput drug screening. The CCK-8 method has low cytotoxicity and can be reused after cell detection. It has better practicability and can replace the MTT method and has a good application prospect.
(6) Alma Blue Method: Alma Blue detection reagent provides a simple, rapid, reliable and safe method for cell proliferation and cytotoxicity detection. Alamar blue exhibits purple-blue non-fluorescence in the oxidized state, and in the reduced state, it is converted into a pink or red fluorescent reduction product, which can be detected by a common spectrophotometer or a fluorometer, absorbance and fluorescence intensity and activity. The number of cells is proportional. Alamar blue is non-toxic and harmless to cells, does not affect cell metabolism, cytokine secretion, antibody synthesis, etc., and can continuously observe and further test the growth state of the same batch of cells.
MTT can be used for all cell types, but MTT is insoluble in standard cell culture media and the formazan crystals it produces need to be dissolved in DMSO. Therefore, MTT is mainly used as an endpoint detection method. The other three salts, like Alma Blue, are soluble and non-toxic. They can be used as a continuous monitoring tool to track dynamic changes in cell proliferation. Among them, XTT is less efficient and needs to add additional factors. WST-1 is more sensitive and effective, and can develop color faster than other salts. The sensitivity of Alamar Blue is also very high, as long as there are 100 cells in the well of the microplate. Tetrazolium salts and Alamar blue redox dyes are very convenient for use in a variety of instruments and high-throughput studies.
2. Detection of ATP content
ATP is a direct source of cellular energy. The ATP content in cells is strictly regulated. Dead cells or cells that are about to die are almost irresorbed by ATP. There is a strict relationship between ATP concentration and cell number measured in cell lysates or extracts. The linear relationship. Therefore, information on cell proliferation can also be obtained by detecting ATP.
ATP detection can be achieved by methods such as color reaction, fluorescence, chemiluminescence or isotope. The most widely used method is based on firefly luciferase (catalyzing luciferin oxidation, consuming ATP. If ATP is present, fluorescein will emit light, the luminescence efficiency is extremely high, and the luminescence amount has a good linear relationship with ATP content. Reflects the amount of ATP in the cells.
3. Live cell fluorescent labeling
Hydroxyfluorescein diacetate succinimide ester (CFSE) is a fluorescent dye that penetrates cell membranes. CFSE can easily penetrate cell membranes, accumulate in living cells and covalently bind to intracellular proteins. The CFSE releases a fluorescent substance, and these covalently bound fluorescent molecules rarely detach from the cells. CFSE-labeled cells can be used for in vivo observation for up to several weeks. When the cells divide, CFSE-labeled fluorescence can be evenly distributed into the two daughter cells, so the fluorescence intensity is half that of the parental cells. Thus, in a proliferating cell population, the fluorescence intensity of each successive cell is logarithmically decreasing, which can be analyzed by flow cytometry at 488 nm excitation and fluorescence detection channels.
4. Enhanced mark detection
Proliferating cells specifically express certain specific proteins, and specific monoclonal antibodies are used to recognize these proliferating cells. For example, in human cells, Ki-67, PCNA, etc. can serve as markers for cell proliferation. However, this method cannot perform high-throughput analysis due to the need for tissue slicing. However, this method is favored by cancer researchers because it can be used to detect the proliferation of tumor cells in vivo.
Beijing Baiou Bowei Biotechnology Co., Ltd. has set up a cell line, which is a center for cell lines. It is specialized in providing cell lines with low generation, short cycle and good activity. With a number of domestic and foreign research and development units, biomedicine, third-party testing institutions, research institutes have a good and stable long-term cooperative relationship! Welcome customers to inquire!
Http://
For Urine, Saliva, water and Any Liquids
Litmus Paper pH Test Strips are a great way to test the acidity or alkalinity of any liquid. Test the pH of your urine or saliva if you are monitoring your alkaline diet or body pH balance. Use them for testing drinking water, kombucha, and any other type of liquid.
Another great use is for testing pool water, hot tubs, aquariums, and spas. Whether you are doing a urinalysis test, testing your body pH or testing the pH of your drinking water, these universal pH strips will do the job.
Ph Test,Ph Strips,Ph Test Strips,Ph Test Kit
Changchun ZYF science and technology CO.,LTD , https://www.zyf-medical.com