Receive a free secondary antibody with your primary antibody purchase. Browse Secondary Antibodies
Boster offers custom polyclonal antibody production for researchers who use non-mammalian models such as Zebrafish, Drophila, C. elegans and Yeast at $600. Contact us for a free consultation.
Boster offers high quality custom antibody production services, including Rabbit and Mouse monoclonal, as well as rabbit polyclonal. For Hu, Mo and Ra targets, we provide Immunoassay development service.For non-hu-mo-ra targets,take advantage of our $600 rare species custom polyclonal program.
Boster provides plate-based multiplex cytokine immunoassay service for analytes from human, mouse and rat. Contact us today and get a free consultation.
Boster offers custom recombinant protein expression service. Available expresssion systems include E. Coli, Yeast, Insect and Mamalian Cells. Get a free consultation today.
Boster offers custom DNA synthesis service for as low as $0.08 per bp. Any gene cloning into any vector, 100% accuracy, Fast turn around time. Get a free consultation today.
Want to have all technical references by your finger tips? Download our FREE eBooks for WB, IHC, ELISA, Flow cytometry and Molecular biology here. These ebooks contain detailed information regarding the principles, protocols troubleshooting tips and optimization tips for their respective assays. Handy for newbies and veterans alike.
Breast Cancer Regulation
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.
• 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.
• 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.
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.
Click the images to enlarge.
Download troubleshooting handbooks for IHC, Western blot and ELISA for FREE.