If the problem is the toxicity of DMSO, you can try to newly set-up your conditions by increasing cell number per well and adding DMSO to the medium, until you reach a good number of colonies in controls, then you can start your treatments.

If you have the possibility, try to dissolve your drugs at the higher concentration and then make intermediate dilutions before adding the drugs to cell medium, in order to reduce the final amount of DMSO.

What's your lowest final DMSO concentration?

1 microliter DMSO/ml should be fine. Make your drug concentrations accordingly (1000 fold higher than final conc.)

Thanks. 5ul of DMSO/2ml. One of the drugs 5FU will not dissolve in lesser volume, I have tried without success.

For many cell lines, 0.25% is too high. In general, try not to exceed 0.1%.

As for 5-FU, methods of many publications indicate that it readily dissolves in water. Have you tried that?

Make sure that your final concentration of DMSO in your working drug solution is not toixic to your cells in cukture.

Correction: it's 2.5ul/ml DMSO. The 5FU I use, I get from Sigma and it needs a solvent to dissolve one of which is DMSO. As my second drug also dissolves in DMSO, I used that for both. I will be very happy to get 5FU which is water-soluble. Do you have a make and product code? Thanks for your suggestions everyone!

Also the 5FU I got will not dissolve in higher concentration, tried and failed.

5-fluorouracil has the same formula and same properties whatever the supplier... It should be water soluble, otherwise it's not 5-FU!

From CAS Database

5-Fluorouracil Chemical Properties,Usage,Production

Chemical Properties

Crystalline

Usage

A potent antineoplastic agent in clinical use. Also an inhibitor of DNA synthesis

Usage

antineoplastic, pyrimidine antimetabolite

Usage

5-Fluoro Uracil is an active metabolite of Doxifluridine (D556750).

General Description

White to nearly white crystalline powder; practically odorless. Used as an anti neoplastic drug, chemosterilant for insects.

Air & Water Reactions

Insoluble in water.

All the supplier I searched have DMSO soluble in DNSO, Methanol, DMF and one may be in hotwater in low concentration. I initially thought it should be water soluble too. That why I asked if anyone couldplease  suggest any source of water soluble 5FU

You should contact Darryl Tong or collaborators, who reported on 5-FU treatment on oral cancer cells (https://www.researchgate.net/publication/12571513_5-Fluorouracil-induced_apoptosis_in_cultured_oral_cancer_cells).

 They dissolved 5-FU in culture medium up to 35 g/L.

Article 5-Fluorouracil-induced apoptosis in cultured oral cancer cells

Here are conditions that worked well for CHO and SIHA colony assays on plastic 6 well plates in our lab:

DMSO concentration should not exceed 0.10%. Each well gets 2ml media. Incubation time is 10-14 days. On Day 6 or 7 check colonies and add 0.5-0.7 mL media to each well carefully. COntrol wells with media only (no DMSO) are good to have. 

Need to determine if there is a DMSO concentration which is non-toxic for the cells you are using, yet sufficiently high to keep your drug in solution.  I would begin by comparing DMSO alone, vs controls (no DMSO).   As the drug likely has limited, yet some water solubility, might bring the drug into solution with DMSO, and dilute it down with medium to its final desired concentration, and then using that final drug/DMSO culture medium preparation at its final concentration.  For a six well plate, probably shouldn't shoot for more than 8-12 well formed colonies per well, so will likely have to use different number of cells per well, depending on the anticipated surviving fraction.

In radiation biology field, DMSO is used as a vehicle (for mock treatment) at a final concentration of 0.1% (i.e., 1 µl/ml) or less, because it scavenges reactive oxygen species.

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