Culturing Cells, the culture conditions used to grow the cells can affect the results and must be taken into consideration when analyzing the data.
Occasionally there may be some particulate material that will not dissolve; this can be removed by filtration or centrifugation. .
The MTT assay involves the conversion of the water soluble MTT bromide) to an insoluble formazan.Mix by vortexing or sonication until dissolved.The age of the cultures, number of passages and details of the growth medium can all be important factors.For non-adherent cells, centrifuge the microplate, pellet the cells, carefully remove as much medium as possible and replace it with 100 L of fresh medium.Our MTT Cell Proliferation Assay Kit provides enough material to perform 1000 individual tests using standard 96-well microplates.Mix the solution gently by inversion or sonication until the SDS dissolves.Each tube makes sufficient solution for 100 tests, using 100 L per well.Add 10 L of the 12 mM MTT stock solution (prepared in step.1) to each well.After labeling the cells with MTT, as described above, remove all but 25 L of medium from the wells.Once prepared, the solution should be used promptly.The result is a sensitive assay with excellent linearity up to approximately 10 6 cells per well.Note that the presence of phenol red in the final assay samples can seriously affect results.Natural variation in the requirements and growth rates of different cell lines make it difficult to provide precise guidelines for preparing your cells.Quick Protocol Option, to shorten the time of the assay it is possible to use dmso (not provided) as a solubilizing agent to dissolve spider man 2 enter electro pc game the formazan.Labeling Cells, for adherent cells, remove the medium and replace it with 100 L of fresh culture medium.2-4 The formazan is then solubilized, and the concentration determined by optical density at 570.Our Vybrant MTT Cell Proliferation Assay Kit provides a simple method for determination of cell number using standard microplate absorbance readers.Incubate at 37C for 4 hours.In general, cells seeded at densities between 5000-10,000 ean golden s4 tutorial dvd cells per well should reach optimal population densities within 48-72 hours.Following the protocol described below, a complete assay requires an overnight incubation.Stored properly, the kit components should remain stable for 12 months.
Materials Required but Not Provided phosphate-buffered saline (PBS sterile HCl,.01 M solution dimethylsulfoxide (dmso) optional.