MTT Cell Proliferation and Cytotoxicity Assay Kit

Description

Catalog #: CSK1156
Kit Components
MTT staining solution 5ml
Formazan diluent solution 55ml
Storage
Store MTT staining solution at -20°C in dark for one year;
Store Formazan diluent solution at room temperature for one year.
Product Description
Alphabioregen’s MTT Cell Proliferation Assay Kit provides a simple method for determination of cell
number using standard microplate absorbance readers. Determination of cell growth rates is widely used
in the testing of drug action, cytotoxic agents and screening other biologically active compounds.
Several methods can be used for such determinations, but indirect approaches using fluorescent or
chromogenic indicators provide the most rapid and large scale assays. Among such procedures, the
MTT assay developed by Mossman1 is still among one of the most versatile and popular assays. The
MTT assay involves the conversion of the water soluble MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) to an insoluble formazan.2-4 The formazan is then solubilized, and the
concentration determined by optical density at 570 nm. The result is a sensitive assay with excellent
linearity up to approximately 96 cells per well.

Protocol
1. Collect logarithmic phase cell, adjust cell suspension concentration; add 100ul floor plate. In
general, cells seeded at densities between 1000-10,000 cells per well (side holes filled with
aseptic PBS buffer).
2. Seed cells in a 5% Co2 incubator at 37℃ until cells bespread well bottom for one floor (cells
number for each well is according to cells’ size and breed speed). Add concentration gradient
drug. Principlely, add drug after cells adhere. 0-10ul per well. Using 3-5 repeating pipettors.
3. Incubate the cells for 16-48 in a 5% Co2 incubator at 37℃, then put upside down under
microscope to observe.
4. Add 10 μl MTT Reagent to each well, continue to culture for 4 hours. If drug react with MMT, you
could centrifugal first then remove nutrient solution. Wash with PBS buffer carefully 2-3 times,
add nutrient solution with MTT.
5. Add 100μl Formazan dilution and shake at low speed for 10min until crystal dissolved
completely. Measure the absorbance at 570nm using an ELISA reader.
6. Meanwhile, set up blank well（culture media, MTT staining solution, Formanzan solution）,
comparation well (cells, drug dissolve media with same concentration, nutrient medium, MTT
staining solution, Formanzan solution)
Note
1. Because detecting by 96 wells, if you need culture cells more than 48h or longer, you should
better saturate the 64 wells in the middle with sterile PBS buffer. In case that, during culture
period, water in side hole evaporates very quickly and nutrient solution or other drug may
condense and the cells situation may become complicated.
2. Before use, please preheat MTT solution at room temperature or 20-25℃ for a moment until it is
thawing.
3. MTT solution should be storage at 4℃ in dark within 2 weeks. For longer tome storage, you
should storage at -20℃, avoid multigelation. Pack it in small package. Once it turns to grayish
green, please stop using it immediately.
4. When Formazan dilution is frozen or appears sediment, you should incubate it at 37℃ water.
Before use it, make it is completely melt or well-distributed mix.
5. Incubate it in dark.
6. MTT is oncogenic and sensitive to bacteria, when using it, please wearing gloves and sterilization