MTT cell proliferation and cytotoxicity detection kit
SB-G4104-500T Size: 500T
* Easy Add MTT & Add Formazan & Measure
* 500 assay measurements per kit
* Optimal measurement at 570nm but work with 560-600nm filter
Description/Introduction:
The full name of MTT is 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (Thiazolyl Blue Tetrazolium Bromide or abbreviated as thiazolyl).
The molecular formula is C18H16BrN5S, and the molecular weight is 414.32.
MTT is often used to detect cytotoxicity and cell proliferation. The detection principle is that the succinate dehydrogenase in the mitochondria of living cells can reduce the exogenous MTT to water-insoluble blue-purple crystal formazan (Formazan). The insoluble formazan will be deposited in the cells, and the amount of the depot can be measured. The cells have no such color reaction without MTT and formazan components in the kit.
Dimethyl sulfoxide (DMSO) is used to dissolve formazan.
The measurement of MTT + Formazan in cells is accomplished by light absorption value with a plate reader. The amount of MTT + crystal formazan formed within a cell number range is proportional to the number of cells. The cells per well must not be confluent or saturated before adding the stimulant or toxic agents. The measurement should be done with cells with cell density near 70% per well.
In the traditional MTT detection method, removing the reaction solution's upper layer containing the MTT substrate is necessary and then add the formazan solution. This step is important for repeatability and reproducibility of the experiment.
Storage and transportation
Transport with wet ice; MTT solution, stored at -20°C and protected from light; formazan dissolving agent, stored at 4°C, protected from light; valid for 12 months.
Component:
Component Number
Component
G4104-500T
G4104-1
MTT solution
5×1 mL
G4104-2
Formazan dissolving agent
50 mL
Product User Manual
1pc
Instructions for use
1. For cell proliferation testing, it is recommended to seed a plate with 100 μL 2000 cells per well; for cytotoxicity experiments, it is recommended to seed a plate with 5000 cells 100 μL per well (adjusted according to cell size and proliferation speed). Culture and transfection or pretreatment for administration according to sentence experiment needs;
2. Add 10 μL of MTT solution to each well and incubate in an incubator for 4 hours;
3. Add 100 uL formazan dissolving agent to the well plate, tap the well plate to mix it, and place it in the incubator to continue incubating for 2-4 h to dissolve the formazan (the incubation time depends on the number of cells. Observe and wait for the formazan to dissolve, then it can be detected);
4. Measure the absorbance of each well at 570 nm. If the 570 nm filter is not available, a 560-600 nm filter can be used.
Precautions
1. If the cell culture time is too long, in order to prevent the liquid from evaporating, it is recommended to fill the outer circle of the 96-well plate with water, PBS or medium to ease the evaporation.
2. In order to reduce the error caused by the experimental operation, it is recommended to set up a duplicate hole to avoid generating bubbles when adding samples or shaking and mixing.
3. The MTT solution is yellow. It should be placed at room temperature or in a water bath at 20-25℃ for a while until it is completely melted before use. If the solution is grayish green, do not use it.
4. If the formazan dissolving agent has precipitated out, it can be dissolved in a water bath at room temperature or 37℃ to promote the dissolution, and then use it after dissolving and mixing.
5. For your health and safety, please wear lab coats and disposable gloves during operation.
