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I am currently testing a series (5-10) of small molecule compounds against an enzyme that are intended as inhibitors. This enzyme is meaningful for cell proliferation. Until now, nothing was active and therefore I could try to test the new molecules in a cytotox assay, to test if they are of any medicinal value. It is known, that these compounds have multiple modes of action and inactivity against the discussed enzyme does not necessarily mean that they are fully inactive.

I am wondering how complicated this is and if you're a beginner on this how long does it take to set up a growth inhibition or viability assay. Currently me gathered experience on protein expression and aspetic technique. But I have no experience with cell culture and sterile bench. My cell lines of interest are melanoma cell lines and or various brest cancer cell lines that are commercially available from multiple known suppliers. Do you think it would be possible to test this in a couple of weeks? I am willing to work 7 days per week if this will speed up the process. What is a realistic time frame to get first results? I would work with commercial inhibitors as control. I have no idea how time intense this process might be and would be interested about your insights.

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The answer to this is, of course, "it depends". There are quite a few factors at play here. As you don't seem to be experienced at cell culture yet, I strongly recommend that you find someone experienced to teach you. It is not something that is easy to pick up, as problems in cell culture (e.g. low-level contamination, cross-contamination, old cells, overgrowth, under-seeding) can be subtle and difficult to identify without experience.

It will take at least 4 weeks for experience to kick in, and cells aren't like bacteria in that they won't grow overnight to suitable levels for sub-culturing/experimentation; it'll take days to weeks of growth from a frozen vial to having enough to work with. This takes time alone. You'll need to be in some weekends anyway; when cells need to be split or fed, that's when you'll be in!

It takes time to learn the characteristics of each cell line, how fast it grows and how it behaves under certain conditions. If you get the conditions wrong, it will invalidate your experiment. Many cell lines will tolerate quite a bit of mishandling (e.g. growing to too high a density), but others will stop growing if you seed at too low a density and/or they become too confluent. This varies from cell line to cell line and even cell lines within a single type (e.g. melanoma) can be different from others.

You also need appropriate media for each cell line - many culture media use phenol-red as an indicator dye, but this is an estrogen analogue, which can affect growth of hormone-sensitive lines (often the case with breast cancer lines for example). Some lines are sensitive to glucose concentration or need a special supplement to grow. Knowing these sorts of things are critical to your experiments and is where experience kicks in. You will need to start by looking up the growth conditions of each cell line you plan to use.

Once you are experienced, it can be a fairly simple process. With enough cells and the right equipment (e.g. repeat dispense function multi-channel pipettes), I can easily set up 20 or more 96-well plates for viability assays (IC50 style) in a day. There are lots of other options on assays too, how long they take and what read-outs (MTT/XTT?, neutral red? cell count? luminescence or fluorescence-based) you use are variables too.

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  • $\begingroup$ Thanks. I am now actually working of these experiments yet. For me, the biological part is not the challenge but the synthesis of Activa compounds. For the viability test I want to use the MTT assay. When it will show some.effect on the cells I want to proceed with apostosis tests and than metabolite studies by LC MS/MS. Keep thumbs pressed that my compounds will kill cancer cells. $\endgroup$
    – raptorlane
    Commented Mar 21 at 18:41
  • $\begingroup$ Just wanted to follow up on this. I am handling 3 different cell lines at the moment. Cells are growing as expected, splitting freezing etc. everything seems to work. But I am not confident with my MTT assay results at the moment. The triplicates ain't looking good and I think it is because of the DMSO step to solubilize the furazan crystals. $\endgroup$
    – raptorlane
    Commented Apr 6 at 21:50

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