Tetrazolium salt (MTT) colorimetric assay
Tetrazolium salt (MTT) colorimetric assay
It is mainly used to measure the level of cellular energy metabolism to indirectly reflect cell proliferation.
Operation method
MTT method
Principle
Succinic dehydrogenase in the mitochondria of living cells reduces exogenous MTT to insoluble blue-violet crystals and deposits them in the cells, whereas dead cells do not have this function. Dimethyl sulfoxide dissolves the purple crystals in the cells, and its light absorption value is measured at 490 nm with an enzyme-linked immunoassay, which can indirectly reflect the cell number. Within a certain cell number range, the amount of MTT crystals formed is proportional to the cell number.
Materials and Instruments
Cells Move I. Inoculation of cells Caveat 1. Select the appropriate cell inoculation concentration. Before the MTT test, each kind of cell should be measured for its adherence rate, doubling time and growth curve under different inoculated cell number conditions, and then determine the number of inoculated cells in each well and the incubation time in the test. 2. Avoid serum interference, generally choose less than 10% fetal bovine serum culture solution for the test. 3. Set up a blank control, and the test parallel to the set of blank control holes without adding cells and only add culture medium. When the final colorimetry is performed, the blank wells are adjusted to zero. For more product details, please visit Aladdin Scientific website.
D-Hanks Liquid Bovine Serum RPMI1640 Dual Antibody 0.08% Trypsin
Purification bench Centrifuge Constant temperature water bath Refrigerator Inverted phase contrast microscope Incubator Straws Glass vials Culture flasks Waste tanks Pipette tips Gun tips Gum plugs Centrifuge tubes Volume spiking gun Red blood cell counting plate
1. Digest monolayer cultured cells with 0.08% trypsin.
2. Prepare a single cell suspension with RPMI1640 culture medium containing 10% fetal bovine serum.
3. Inoculate the cells in 96-well culture plates at 103-104 cells per well with a volume of 200 ul per well.
II. Cultured cells
1. Transfer the culture plate into a CO2 incubator.
2. Incubate at 37℃, 5% CO2 and saturated humidity. (The incubation time depends on the purpose and requirements of the experiment)
III. Color presentation
1. 20 ul of MTT solution (5 mg/ml) was added to each well, and incubation was continued for 4 h at 37℃ in an incubator.
2. Terminate the incubation and carefully aspirate the culture supernatant from the wells.
3. For cells grown in suspension, centrifuge (1000 rpm, 5 min) and discard the culture medium from the wells.
4. Add 150 ul DMSO to each well and shake for 10 min to fully dissolve the crystals.
IV. Colorimetry
1. Select the wavelength of 490 nm, determine the light absorption value of each well on the enzyme immunoassay detector, and record the results.
2. Plot the cell growth curve with time as the horizontal axis and light absorption value as the final axis.