Does anybody know what is the highest concentration of a Stock solution of MTT (powder from Sigma ) in Culture Medium ?
I know from Sigma that they tested their powder up to 5mg/ mL and that in the handbook the solubility in water reaches 10mg/mL . But does anyone with high competences in chemistry or by experience know wheter I can dilute it also in Medium up to 10 mg/mL ?!
And do you filter it before use?! I would be very grateful for any precise explanation !
Thanks !
Hi Vanessa, may I ask, why would you need it to be 10mg/1ml instead of 5mg/ml. . For me, i used to use PBS not water to dissolve it in 5mg/ml , then cover tube with foil because it is light sensitive and perform vigorous and almost 10 minutes shaking with vortex ,then use 0.2 μm filter to filter it. it took me a while to get the absorbance reading between 0.1-1, i had to reduce the number of seeded cells, so make sure they are between 0.1-1 , especially when you are using powder not a complete kit. good luck
I wanted to prepare a higher Stock to reduce the amount of basic Medium or PBS added to the medium, to not dissolve molecules like aa or glucose to much .....
Are you sure that tha absorbance has to be between 0.1 - 1? I was thaught that newer machines are able to measure exact also in higher ranges up to 2 or 3 .....
Thanks!
Dear Vanessa,
Normally In cytotoxicity assays we are using 5mg/ml of mtt as a stock. Dissolving solvent is PBS not to media ( becoz media interferes absorbency of final O.D). If u want to reduce the MTT dissolved PBS then you could add 5 ul (microlitre) per well sufficient.
Before added to the well you should cover the foil or to take amber color tube to preparing after that filter it with o.2 to 2 um filter it
Finally make sure the seeded cells should be 5000 at the time of seeding the cells in the 96 well plate then will get the absorbency has to be in between 0.1 to 1 O.D.
Absorbency depends upon the 1) Number of cells seeded to the plate
2) type of test compound
All the best and do the culture with contamination free
If you use medium, use phenol free.
Usually speaking, MTT solubility in water is 10 mg/ml, in PBS or media is 5 mg/ml, and in ethanol is 20 mg/ml.
For cytotoxicity assay, as Srinivas said above, we also use 5 mg/ml in PBS, filtered, stored at 4C for frequent use (within 3 days). If you want to store longer, make small aliquots and store at -20C.