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What are the steps of the MTT lab, and what are the caveats
MTT is usually used to determine the cytotoxicity of drugs (including other treatments such as radiation irradiation) in vitro as well as cell proliferation and cell activity. This method has been widely used to detect the activity of some bioactive factors, large-scale screening of anti-tumor drugs, cytotoxicity test and radiosensitivity test of tumor, and so on. It is characterized by high sensitivity and economy. However, because the product of MTT produced by reduction is insoluble in water, it needs to be dissolved before it can be detected. This not only increases the workload, but also affects the accuracy of the experimental results, and the organic solvent dissolved in α-Zan also damages the experimenter. In MTT experimental steps should be noted that MTT is carcinogenic, be careful when used, it is best to wear that transparent film gloves. The MTT needs to be aseptic, and MTT is sensitive to bacteria; it doesn't matter to add light to the 96-well plate. After all, the time is short, or you can turn off the lights on the console when you're not at ease.
MTT Experimental steps-Adhesion cells
1. The logarithmic phase cells were collected and the cell suspension concentration was adjusted. 100uls were added to each hole. The cell density to be measured was adjusted to 1000 × 10 000 holes (filled with aseptic PBS at the edge of the hole).
2.5% CO _ 2, incubated at 37 ℃, to the bottom of the cell monolayer covered with holes (96 holes flat floor), adding a drug of concentration gradient, in principle, after the cell adheres to the wall can be added, or two hours, or half a day, but we often lay the board the afternoon before the day. Take the medicine the next morning. Generally 5-7 gradients, 100 ulls per hole, 3-5 multiple holes. 5 are recommended, otherwise it is difficult to respond to the real situation
3.5% CO 2, incubated at 37 ℃ for 16 h for 48 h, observed under inverted microscope.
4 h. If the drug can react with MTT, it can be centrifuged and then discarded, then washed with PBS for 2 times for 3 times, and then added with the medium containing MTT.
5, stop the culture, carefully absorb the medium in the pore.
At 6, 150ul dimethyl sulfoxide was added to each hole and shaking in a shaking bed at low speed for 10 minutes to make the crystalline fully dissolved. The absorbance value of each hole was measured by enzyme-linked immunosorbent assay (OD490nm).
7. At the same time set zero pore (medium, MTT, dimethyl sulfoxide), control pore (cell, drug dissolution medium of the same concentration, culture medium, MTT, dimethyl sulfoxide)
Step of MTT experiment-suspension cells
1. The logarithmic phase cells were collected and the concentration of cell suspension was adjusted to 1 × 10 ~ 6 ml. The 1640 (serum-free) medium 40ul was supplemented by 1 in sequence. (2) dilution of 10ul with Actinomycin D () lg/ml, should be pretested to find the best dilution (1 / 10 / 1 / 20), (3) 10 uls should be detected; 4Cell suspension 50ul (5 × 104cell/ hole), total 100ul added to 96-well plate (edge hole filled with aseptic water). Control each plate (add 100% (storage liquid 100 1640).
2, incubated with 5%CO2 for 16 hours at 37 ℃ for 48 hours, and observed under inverted microscope.
(3) 10 ul MTT solution (5 mg/ml, 0.5%MTT) was added to each hole and cultured for 4 h. (suspension cells are recommended to be cultured with WST-1, for 4 hours and skip step 4). Direct enzyme-linked immunosorbent assay OD570nm (630nm calibration) is used to measure the absorbance of each hole.
4, centrifuge (1000 rpm x10min), absorb the supernatant carefully, add 100 ul dimethyl sulfoxide to each hole, shake the 10min at low speed on the shaking bed, make the crystal dissolve fully. The absorbance of each hole was measured by enzyme-linked immunosorbent assay (OD570nm) (630nm calibration).
At the same time, the zero-setting pore (medium, MTT, dimethyl sulfoxide) and the control pore (cell, the same concentration of drug dissolution medium, culture medium, MTT, dimethyl sulfoxide) were set at the same time, and 3 repores were set in each group.
It is important to note that the MTT method can only be used to detect the relative number and relative viability of cells, but not the absolute number of cells in the MTT procedure. In order to ensure the linearity of the experimental results, the best absorbance of MTT is in the range of 0 ~ 0. 7 when the results are detected by the enzyme marker. In the preparation of MTT, PBS was used to dissolve, and some were mixed with saline, and 60 ℃ water bath was used to assist in solubilization.
The material comes from the Internet.
To learn more about biochemical reagents, please follow the official website: http://www.hnhbsj.com
(all content of this website, indicating that the source is "Reagent", the copyright is owned by the Reagent, without authorization, no media, websites or individuals may be reproduced, otherwise, legal liability will be investigated, Authorization shall be reproduced with a reference to "Source: foreign Reagent". This website indicates that the source of other media content for reprint, reprint only for opinion sharing, copyright belongs to the original author, if there is copyright infringement, please contact us in time.)
MTT Experimental steps-Adhesion cells
1. The logarithmic phase cells were collected and the cell suspension concentration was adjusted. 100uls were added to each hole. The cell density to be measured was adjusted to 1000 × 10 000 holes (filled with aseptic PBS at the edge of the hole).
2.5% CO _ 2, incubated at 37 ℃, to the bottom of the cell monolayer covered with holes (96 holes flat floor), adding a drug of concentration gradient, in principle, after the cell adheres to the wall can be added, or two hours, or half a day, but we often lay the board the afternoon before the day. Take the medicine the next morning. Generally 5-7 gradients, 100 ulls per hole, 3-5 multiple holes. 5 are recommended, otherwise it is difficult to respond to the real situation
3.5% CO 2, incubated at 37 ℃ for 16 h for 48 h, observed under inverted microscope.
4 h. If the drug can react with MTT, it can be centrifuged and then discarded, then washed with PBS for 2 times for 3 times, and then added with the medium containing MTT.
5, stop the culture, carefully absorb the medium in the pore.
At 6, 150ul dimethyl sulfoxide was added to each hole and shaking in a shaking bed at low speed for 10 minutes to make the crystalline fully dissolved. The absorbance value of each hole was measured by enzyme-linked immunosorbent assay (OD490nm).
7. At the same time set zero pore (medium, MTT, dimethyl sulfoxide), control pore (cell, drug dissolution medium of the same concentration, culture medium, MTT, dimethyl sulfoxide)
Step of MTT experiment-suspension cells
1. The logarithmic phase cells were collected and the concentration of cell suspension was adjusted to 1 × 10 ~ 6 ml. The 1640 (serum-free) medium 40ul was supplemented by 1 in sequence. (2) dilution of 10ul with Actinomycin D () lg/ml, should be pretested to find the best dilution (1 / 10 / 1 / 20), (3) 10 uls should be detected; 4Cell suspension 50ul (5 × 104cell/ hole), total 100ul added to 96-well plate (edge hole filled with aseptic water). Control each plate (add 100% (storage liquid 100 1640).
2, incubated with 5%CO2 for 16 hours at 37 ℃ for 48 hours, and observed under inverted microscope.
(3) 10 ul MTT solution (5 mg/ml, 0.5%MTT) was added to each hole and cultured for 4 h. (suspension cells are recommended to be cultured with WST-1, for 4 hours and skip step 4). Direct enzyme-linked immunosorbent assay OD570nm (630nm calibration) is used to measure the absorbance of each hole.
4, centrifuge (1000 rpm x10min), absorb the supernatant carefully, add 100 ul dimethyl sulfoxide to each hole, shake the 10min at low speed on the shaking bed, make the crystal dissolve fully. The absorbance of each hole was measured by enzyme-linked immunosorbent assay (OD570nm) (630nm calibration).
At the same time, the zero-setting pore (medium, MTT, dimethyl sulfoxide) and the control pore (cell, the same concentration of drug dissolution medium, culture medium, MTT, dimethyl sulfoxide) were set at the same time, and 3 repores were set in each group.
It is important to note that the MTT method can only be used to detect the relative number and relative viability of cells, but not the absolute number of cells in the MTT procedure. In order to ensure the linearity of the experimental results, the best absorbance of MTT is in the range of 0 ~ 0. 7 when the results are detected by the enzyme marker. In the preparation of MTT, PBS was used to dissolve, and some were mixed with saline, and 60 ℃ water bath was used to assist in solubilization.
The material comes from the Internet.
To learn more about biochemical reagents, please follow the official website: http://www.hnhbsj.com
(all content of this website, indicating that the source is "Reagent", the copyright is owned by the Reagent, without authorization, no media, websites or individuals may be reproduced, otherwise, legal liability will be investigated, Authorization shall be reproduced with a reference to "Source: foreign Reagent". This website indicates that the source of other media content for reprint, reprint only for opinion sharing, copyright belongs to the original author, if there is copyright infringement, please contact us in time.)
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