How do you make a MTT solution?
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How do you make a MTT solution?
Prepare a 12 mm stock solution by dissolving 5 mg of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] in 1 mL of PBS. This stock solution will be enough for 100 reactions (10 µL per reaction). Once prepared, the MTT solution can be stored for 4 wk at 4°C protected from light.
How do you dilute MTT powder?
Prepare MTT solution MTT is soluble in water (10 mg/mL), ethanol (20 mg/mL), and buffered salt solutions and culture media (5 mg/mL). We recommend using a 5 mg/mL solution in PBS. Mix by vortexing or sonication.
What is the detergent for MTT assay?
detergent sodium dodecyl sulfate
In the MTT assay a solubilization solution (dimethyl sulfoxide or acidified ethanol solution, or a solution of the detergent sodium dodecyl sulfate in diluted hydrochloric acid) is added to dissolve the insoluble purple formazan product into a colored solution.
How much DMSO added in MTT assay?
Add 100 μl of DMSO to each well and mix by pipetting up and down. Note: Be sure to add 100 μl DMSO to a well without cells as a blank.
How do I improve my MTT assay?
Using freshly prepared FILTERED MTT (0.5mg/ml concentration) can improve results. Secondly, after 4 hrs incubation with MTT, remove MTT by multichannel pipette, and then add DMSO to dissolve formazon (100microlitre ) in each well and take readings instantly.
Does MTT react with DMSO?
A reaction occurs between medium and a solution of MTT formazan in DMSO which changes the shape of the absorbance spectrum of the solution. The resulting optical density is not however greatly dependent upon the volume of added medium in the range 1-10 microliters.
Should I remove MTT before adding DMSO?
Yes, you have to remove the MTT medium before adding DMSO. To be sure you have to revise all the official protocol of MTT assay. It is better using the related compund WST-1, or any of its upgrades. This has better signal-to-noise ratio, and you do not have to solubilize the formazan crystals by adding DMSO!
Why is DMSO added in MTT?
DMSO is added at the end of the reaction to dissolve the formazan crystals formed from the reaction. Otherwise, you can not properly measure the color density of the reaction. DMSO is added only after incubation with MTT dye, after you remove the medium from cells, in order to dissolve formazan crystals.
