Hello Reyes.

Sorry for late answer.

No, it does not.

Yes, that's correct. Often when you thaw the frozen stock aliquot solution, they would have further precipitations generated by freezing and thawing procedure and probably some particles present in the tube from the manufacturer. Therefore you need to the same centrifugation and supernatant to be added to the cell culture.

If the particles are present in the culture, they would distort the result due to the non-specific toxic effects or engulfment by the cells.

Best wishes.

Hello Reyes.

Make the highest stock solutions possible of the all of the contents of the two chemicals in absolute ethanol separately.

For example, ~400-500 mg/ml (just estimates) each in ethanol (100% EtOH). Dissolve them completely. To make them amenable to the following step for cell culture, dilute 2x with autoclaved ddH2O (now ~200-250mg/ml final stock solutions; 50% EtOH).

Some of the components may well be precipitated. Spin them at 12,000rpm in Epen tubes take only clear supernatant into new tubes and discard the precipitates in tubes. The concentrations of the chemical are considered as the original amount since precipitation will be continuously occurring when added to culture medium.

It is good enough to describe the procedure exactly how you prepared the stock sol.

When you use for culture, dilute 10X the final stock sol (1st dilution, 10x dilution) with culture medium (~20-25mg/ml; 10% EtOH). You may need spin down similarly.

Add these first-diluted stock sol into culture dish medium, say 10ul into 1ml culture medium (2nd dilution, 100X dilution: 0.1% EtOH ) to make final concentrations of 200~250ug/ml) to be tested. Other concentrations can be tested by adding appropriate volumes of 1st-diluted stock sol. The EtOH would evaporate in the medium. All stock sols in 50% EtOH are aliquoted and stored at -70C in tight capped Epen tubes for years, Throw away the remaining solution once the stock solutions are thawed.

Good luck

Dear Prof. Sang Ho Lee,

Thank you very much for your help. Just one more doubt: Does the EtOH affect the Polyphenol's stability or state of oxidation?

The stock solutions are aliquoted and stored AFTER the spin+precipitares discarding procedure,right?

I am very pleased about your message

Hello Reyes.

Sorry for late answer.

No, it does not.

Yes, that's correct. Often when you thaw the frozen stock aliquot solution, they would have further precipitations generated by freezing and thawing procedure and probably some particles present in the tube from the manufacturer. Therefore you need to the same centrifugation and supernatant to be added to the cell culture.

If the particles are present in the culture, they would distort the result due to the non-specific toxic effects or engulfment by the cells.

Best wishes.