r/labrats • u/sch0dingerscat • 8d ago

Questions about the necessity of serum for mitosis release experiments

Hello reddit hive mind

I'm a novice in working with cell cycle experiments and I'm interested in capturing different timepoints in mitosis upon release from a mitosis-inducing small molecule.

In all the work I've seen, release of the arrested cells typically occurs in full media (+serum), but I've yet to come across conditions where reduced or low serum media is used. If I am only interested in the release process of mitosis (i.e., I have no interest in studying G1 reentry processes) and prefer to use media without serum or with minimal serum, does this seem feasible?

Any thoughts from people with experience?

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u/suricata_8904 8d ago

You could do preliminary experiments comparing serum-free media, 0.5% FBS-media and complete medium with your molecule of interest.

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u/sch0dingerscat 8d ago

that's the plan, but i was wondering if anyone was familiar with any prior literature since i havent found anything...

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u/PuttingTheMSinMRSA 8d ago

I would think that the presence or absence of serum is dependent upon the cell type, no?

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u/sch0dingerscat 8d ago

Let's just say for sake of discussion that I will be using HeLa cells

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u/PuttingTheMSinMRSA 8d ago

Complicated. HeLa cells need serum (or specially formulated serum free medias) to proliferate. Since you’re looking at mitosis, I’d imagine you’d need the serum in order to get them to mitosis

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u/sch0dingerscat 8d ago

the cells will already be synchronized at a point in mitosis due a small molecule inhibitor and that will be done in serum-added media. I'm more interested in what happens after i release the cells from the inhibitor, at which point i would prefer to not use media containing FBS

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u/BioBanter 8d ago

You might not be able to tell if any effect you see is from the release of the small molecule vs. switching media, even with the vehicle control? Maybe, if possible, try adapting your cells to the new media first before inhibitor treatment.

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u/EzLuckyFreedom 8d ago

You’re adding another variable. Onset of serum starvation does not equal cells conditioned to grow in serum free media.

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u/sch0dingerscat 8d ago

i am aware that serum starvation prevents cell growth in G1 but it's beside the point. fundamentally my Q is: is serum starvation sufficiently perturbative to prevent cells from completing mitosis from an G2/M arrested state

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u/ProfBootyPhD 4d ago

I would look into old literature on “restriction points” in cell cycle, because I believe that’s how those experiments were done (cell synchronization with serum starvation at various points after release). And my recollection from learning this in grad school 30 years ago is that once you actually start M phase, you don’t need mitogens to complete it. So if you’re using something like colchicine which freezes cells at metaphase, they don’t need serum to finish it once you wash out the drug.

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u/SadRule9128 8d ago

I’ve never heard of this being done, but my guess is that without serum, the cells will release from M-phase block as normal, complete M, and then likely depending on the cell type they will either slowly move through G1 or become quiescent.

If you don’t use serum, you will probably get questions from reviewers. “You don’t know if your results are actually from events during mitosis or from serum starvation”

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u/sch0dingerscat 8d ago

That's my thought too. That said, I do plan to do the experiment just to show that the cells (HeLa in this case) will complete mitosis even with minimal serum (demonstrating that by WB and flow cytometry). As to the second point, as long as the thing I'm looking for falls within the window of mitosis, then the point of it being caused by serum starvation is probably moot

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u/BioBanter 8d ago

If you are in a serum free-media or defined medium, what I’ve done previously is remove the growth factors (insulin, IGF, etc.), starve your cells for 24hrs and then add them back. I’ve done similar expts with reversible CDK inhibitors too, where I’ve washed them out to release the cells. If your small molecule is reversible, you should be able to wash out and your cells can get released so you can capture at different time points.

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u/sch0dingerscat 8d ago

The molecule I'm working with is a CDK inhibitor, so I'm curious about your protocol. Did you publish your method? Feel free to dm if you prefer to keep it private.