MTT Cell Proliferation and Cytotoxicity Assay Kit

Instruction manual

First Edition (Revised on April, 2016)

[PRODUCT INFORMATION]

The tests of cell proliferation and cell toxicology, it was widely used to evaluate some factors that affected the cell cycle, such as growth factors, cytokines, mitogens, and drugs that affect cell division and proliferation, ect. It had become an important tool in medical testing, drug testing and food and environmental safety testing.

[PRINCIPLE]

Methylthiazolyldiphenyl-tetrazolium bromide (MTT) is a compound dye that accepts hydrogen ions with a positive charge and a strong membrane permeability. When MTT enters living cells, mitochondrial succinate dehydrogenase reducted MTT to a deep blue formazan crystal MTT. Formazan crystal does not have cell membrane permeability, will be permanently preserved in living cells, color the living cells. While dead cells, on the one hand, cell membranes are disrupted  have no selective permeability, on the other hand, does not have the ability of reduction MTT to formazan crystal, will not be colored. Formazan crystal has the highest absorption peak at the wavelength of 570nm, OD_570nm is detected via enzyme-labeled instrument，it's a good response the reaction system of the formazan crystal production. The number of generation of formazan crystal and living cells is proportional , used to evaluate cell viability and proliferative status.

[REAGENTSAND MATERIALS PROVIDED] 

Specification	1000 tests
Reagent A: MTT (powder)	50 mg
Reagent B: MTT solution	10 ml
Reagent C: Formazan solution	120 ml
Literature	1

[STORAGEAND PERIOD OFVALIDITY]

Reagent A and reagent B should be preserved at -20℃，reagent C is stored at 4℃. Unopened reagents，valid for 12 months.

[APPLICATIONS] 

A．The Preparation of Reagents Before Experiment

1. MTT solution configuration

25mg MTT powder was directly dissolved in 5ml MTT solution, the working concentration is formulated as 5mg/ml, filtratingand removing bacteria. The solution can be stored at 4℃ for a short time(the shorter the better). It is suggested that after dissolving the solution. Direct pack the remaining liquid in small doses, -20℃ preservation, it can be kept for 3-6 months, avoid repeated freezing and thawing.

2. The solution of formazan

The solution of formazan may be deposited at low temperature or at room temperature, only need 37℃ water bath to make it fully dissolved, use after mixing.

B．MTT Cell Proliferation Assay

1. Inoculate cell：Cell suspension was prepared with  10% FBS, adjust the cell density, 5×10³~2×10⁴ cells per well were inoculated to the 96 plate, every well volume is 100µl, set the zero adjustment well at the same time(culture medium +MTT.+the solution of formazan), the control well(cell+ dissolved medium of the same concentration drug +culture medium +MTT+ the solution of formazan).

2. 37℃, 5% CO₂, culture 24-72h.

3. Optional: For adherent cell, remove old cultures, exchange fresh culture medium 100µl per well（phenol red free）; for suspension cells, centrifuge 96 wells plate, remove as many supernatant as possible, then add fresh culture medium 100µl per well（phenol red free）, to suspension cells.

4. Add 10µl the working concentration of MTT(5mg/ml).

5. After low speed mixing on the level table for 1min, incubate in 37℃ for 4 hours.Mind：for high-density cells(>100,000 per well), the incubation time can short to 2 hours.

6. For adherent cell, direct suck out the old medium, avoid destroying the single cell layer. For suspension cells, centrifugal to collect cells, remove culture medium.

7. Add formazan solution 100µl per well, mix fully with gun, then incubate in 37℃ for 4 hours.

8. After incubation, place in enzyme-labeled instrument and shake up, select 570nm, determin the absorbance.

[REAGENT PREPARATION]

1. The MTT solution is sensitive to temperature, light and microbial environment, preservation for a long time requires low temperature, shading and sterile condition. Do not use when the solution color is green.

2. The MTT solution solidifies at low temperature, please keep it at room temperature or 20~25℃, being use until the solution is completely dissolved and mixed.

3. If use the 96T plate to culture and detect cells for a long time, to prevent the evaporation of liquids make the test results instable, you can fill with sterile PBS, water or culture medium in the edge of plate.

4. Add the formazan solution in the plate, to observe the dissolution of formazan solution in ordinary optical microscope, when the formazan was dissolved to detect in OD_570nm is most accurate, prolonged incubation will decrease the detection of sensitivity. Usually incubate at 37℃ for about 4 hours, purple crystals will dissolve completely. If the purple crystals are smaller and less, the dissolution time will be shorter. If the purple crystals are larger and more, the dissolution time will be longer.

5. The MTT solution has weak toxicity, the formazan solution is caustic, for your safety and healthy, please wear  lab coat and disposable gloves and do experiments in accordance with the laboratory standard operating manual.