MTT Cell Proliferation and Cytotoxicity Assay Kit
|Pack size||1 kit (for 500 assays)|
|Applications||Measurement of cell proliferation in response to growth factors, cytokines and nutrients.|
Measurement of cytotoxicity
To study cell activation
List of Components
|MTT labeling reagent||1||5mL||5mg/ml in PBS||AR1156-1|
|Formazan solubilization solution||1||55mL||10% SDS in 0.01 M HCl||AR1156-2|
|Product Name||MTT Cell Proliferation and Cytotoxicity Assay Kit|
|Pack Size||1 kit (for 500 assays)|
|Storage & Expiration||Upon receipt store MTT labeling reagent at -20°C. Protect from light. It is stable at -20°C for one year.
Store Formazan solubilization solution at room temperature. It is stable at room temperature for one year.
|Equivalent||Thermofisher (Product No. V13154), Millipore Sigma (Product No. 11 465 007 001)|
|For Use With (Equipment)||Microplate Reader|
|Label or Dye||MTT|
|Description||Boster’s MTT Cell Proliferation Assay Kit provides a simple method for determination of cell number using standard microplate absorbance readers.|
|Cite This Product||MTT Cell Proliferation Assay Kit (Boster Biological Technology, Pleasanton CA, USA, Catalog # AR1156)|
|Application||Measurement of cell proliferation in response to growth factors, cytokines and nutrients.
Measurement of cytotoxicity
To study cell activation
*Our Boster Guarantee covers the use of this product in the above tested applications.
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. The non-radioactive, colorimetric assay system using MTT was first described by Mosmann. The assay is designed for the spectrophotometric quantification of cell growth and viability without the use of radioactive isotopes. The MTT assay involves the conversion of the water soluble MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to an insoluble purple formazan crystals. The formazan crystals formed are then solubilized, and the concentration of resulting colored solution determined by optical density at 570 nm. The result is a sensitive assay with excellent linearity up to approximately 106 cells per well.
Important Product Information
• The culture conditions used to grow the cells can affect the results and must be taken into consideration when analyzing the data. The age of the cultures, number of passages and details of the growth medium can all be important factors.
• For Formazan solubilization solution, frozen block or precipitates may form during shipment or storage, in which case the container should be warmed to +37°C and thoroughly mixed.
• After thawing, the MTT labeling reagent may be stored protected from light at 4°C for up to 4 weeks, in which case a sterile filtration of the reagent is recommended.
• The presence of phenol red in the final assay samples can seriously affect results. We recommend that the cells be cultured in medium free of phenol red, if possible.
• MTT is oncogenic. Please wear gloves to operate.
Additional Materials Required
• 10μL, 100-200μL and multi-channel pipettes
• 96 well plate
• CO2 incubator
• Plate reader capable of reading absorbance at 570nm
1. Collect cells at logarithmic phase, count the cells.
Note: Add 2000 cells per well for cell proliferation assay and 5000 cells per well for cell cytotoxicity assay. Cells number of each well depends on cell density and growth rate.
2. For adherent cells, remove the medium and replace it with 100 µL of fresh culture medium. For non-adherent cells, centrifuge the 96 well microplate, pellet the cells, carefully remove as much medium as possible and replace it with 100 µL of fresh medium.
3. Add 10 μL of the MTT labeling reagent to each well. Include a negative control of 10μL of the MTT labeling reagent added to 100 µL of medium alone.
4. Incubate the microplate at 37°C for 4 hours in a humidified chamber (5% CO2 ). At high cell densities (>100,000 cells per well) the incubation time can be shortened to 2 hours.
5. Add 100 μL of Formazan solubilization solution to each well and mix thoroughly.
6. Incubate the microplate at 37°C for 4-18 hours in a humidified chamber (5% CO2 ).
7.Check for complete solubilization of the purple formazan crystals and measure the spectropho- tometrical absorbance of the samples using a microplate reader at 570 nm.
Note: The wavelength between 560 and 600 nm can be chosen if 570nm is unavailable.
Quick Protocol Option
To shorten the time of the assay it is possible to use DMSO (not provided) as a solubilizing agent to dissolve the formazan.
1. After labeling the cells with MTT, as described above, remove all but 25 µL of medium from the wells. For non-adherent cells it may be necessary to first centrifuge the plates to sediment the cells.
2. Add 50 µL of DMSO to each well and mix thoroughly with the pipette.
3. Incubate at 37°C for 10 minutes.
4. Check for complete solubilization of the purple formazan crystals and measure the spectropho- tometrical absorbance of the samples using a microplate reader at 540 nm.
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Q: Would there be any issues adding Glutamine in the media when using AR1156?A: Glutamine in the media will not affect the whole experiment.
Q: Will using DMSO to solubilize the formazan crystals affect the cells since it is toxic at high concentrations?A: DMSO will not affect the cells.