Hello Areej Alshamer,

Funalenone is soluble in DMSO. Make a stock solution of Funalenone of a higher concentration say 100mM in 100%DMSO, and make a series of dilutions from the stock (say 1nM to 100uM) using culture media or aqueous buffer. Please note that the final concentration of DMSO when you add Funalenone to the cells should be less than or equal to 0.1% DMSO in order to avoid toxicity caused to the cells by DMSO. Treat the cells with at least five dilutions of Funalenone.

If you need the MTT protocol, I have provided one below.

1. Plate the MCF-7 in a 96-well plate format at a desired cell density such that they are 80% confluent on the day of readout. If cells are overconfluent on the final day of the assay, you may end up with inaccurate results. So, optimize the seeding density for the 96-well plate format before you perform the MTT assay.

2. After 24 hours, when the cells have attached to the substratum, treat the cells with Funalenone by changing the media with treatment media at various concentration ranging from 1nM to 100uM (at least 5 concentrations).

3. Incubate the cells with the treatment media for 24, 48 and 72 hours.

4. After the treatment period, aspirate the treatment media and add MTT reagent to a final concentration of 0.2 to 0.5mg/ml and incubate in the dark for 1 to 4 hours for the formation of formazan crystals.

5. After incubation, solubilize the crystals into a colored solution with the solubilizing solution which may include either dimethyl sulfoxide or acidified isopropanol solution, or a solution of 10% SDS in 0.01M hydrochloric acid.

6. Measure the absorbance at a wavelength of 570 nm.

7. IC50 value may be calculated from the graph constructed by plotting cell survival versus Funalenone concentration.

Please note that IC50 stands for half-maximal inhibitory concentration, and it's a measure of how much of a drug is needed to inhibit a biological process by half. It is a widely used and informative way to measure a drug's efficacy and potency.

Good Luck!