Hi Shakti,

SERUM STARVATION is always the "Prerequisite step" for all kind of cell based assay; for instances - migration, invasion, soft agar colony formation, proliferation, GTPases assay, kinase assay or even sometime for specific phosphorylation or dephosphorylation assays.

When you serum starve the cells you need to keep it for at least one doubling time of your cells (depend on cell-lines). 2h is too low, and it is usually O/N.

What it DOES ?-- Since we do not put any growth factors (in serum free media), cells do not receive any signal for division so undergo quiescent phase (G0) and wait for signal. Since all cells are in different phase we need longer time to bring them to the same line on a racing track. Now they respond to your treatment uniformly and show up enriched effect.

However, only serum starvation is not sufficient for cell synchronization since cells itself secrete some amount of growth factors. So for my routine experiments I use reversible inhibitors in serum free media (for critical experiments).

"Nocodazole" is one of the best studied and widely used reversible reagent for microtubule polymerization inhibition and hence arrest cells in G2-M phase (at prometaphase) and synchronize at this phase very effectively (>80% cells).

For my experiments I use nocodazole 50ng/mL of serum free media for 18h. I have checked the cell cycle profile by FACS in both conditions (serum starvation and nocodazole treatment) and saw the difference. [You can also extent it to 24h, but need standardization].

NOTE: Cells after nocodazole treatment look rounded under microscope (because they are arrested in metaphase), but attached to plate.

Nevertheless, you can also use other inhibitors depends on at which phase you want to arrest/synchronize the cells.

Good Luck.

Hi Shakti,

SERUM STARVATION is always the "Prerequisite step" for all kind of cell based assay; for instances - migration, invasion, soft agar colony formation, proliferation, GTPases assay, kinase assay or even sometime for specific phosphorylation or dephosphorylation assays.

When you serum starve the cells you need to keep it for at least one doubling time of your cells (depend on cell-lines). 2h is too low, and it is usually O/N.

What it DOES ?-- Since we do not put any growth factors (in serum free media), cells do not receive any signal for division so undergo quiescent phase (G0) and wait for signal. Since all cells are in different phase we need longer time to bring them to the same line on a racing track. Now they respond to your treatment uniformly and show up enriched effect.

However, only serum starvation is not sufficient for cell synchronization since cells itself secrete some amount of growth factors. So for my routine experiments I use reversible inhibitors in serum free media (for critical experiments).

"Nocodazole" is one of the best studied and widely used reversible reagent for microtubule polymerization inhibition and hence arrest cells in G2-M phase (at prometaphase) and synchronize at this phase very effectively (>80% cells).

For my experiments I use nocodazole 50ng/mL of serum free media for 18h. I have checked the cell cycle profile by FACS in both conditions (serum starvation and nocodazole treatment) and saw the difference. [You can also extent it to 24h, but need standardization].

NOTE: Cells after nocodazole treatment look rounded under microscope (because they are arrested in metaphase), but attached to plate.

Nevertheless, you can also use other inhibitors depends on at which phase you want to arrest/synchronize the cells.

Good Luck.

Serum starvation is an easy way to synchronize your cells.  By stalling them at G0 you can ensure that all the cells start at the same cell cycle position.  This then can remove variation in your assays, such as growth an signalling impacts which can be caused by cells in different cell cycle phases.

Hello Indrajit Bhai,

Thank you so much for the informative answer. Earlier I have used RO3306 for synchronizing the cells at G2/M border for some study on CDKs but I could not understand that how only serum starvation can do the job. How can it bring all the cells to a starting point?

Thank you for clearing my doubt. Thank you so much.

Regards.

Hi Alex,

How can one be sure that all the cells or almost all the cells are at the same beginning point after synchronization using only the serum starve condition?

Please put some light on it.

Hi,

Cell synchronization is an important prerequisite for several applications. The method used depends on what cell-cycle stage that we what the cells to be synchronized at. Serum starvation sure depletes the nutrient availability and hence maybe this is the reason that cells are arrested at G0 stage as a response to serum deprivation. Also, cell lines or cells in cultures can be synchronized at the G2 stage using methotrexate. The removal of  methotrexate would enable one to obtain cells at the S phase. The most well known blocker at the M phase is colchicne which is the most commonly used spindle-blocker that is used for cytogenetic analysis.

Hence, the cell cycle stage at which we want the cells to be synchronized will require a different cell-cycle stage blocker. This in turn depends on the applications of such synchronized cells.

Hi Shakti,

whatever you do to synchronize your cells in the end, cell cycle analysis by PI staining and FACS analysis will be the easiest way to check if you succeeded. Compared to non-synchronized cells you should then find the majority of your cells in the phase you blocked them in.

Also beware that cell cycle blocking agents will be toxic if incubated for too long, so you might want to titrate the optimal timing by running a time course and FACS analysis.

Hope that helps.

Dear Shakti,

since you are interested in cancer cells, serum deprivation will not work as well as it works for primary or other non-cancerous cell lines. Cancer cells may still cycle in the absence of growth stimuli.

Are you interested in a particular cell cycle stage?

double thymidine block (DTB), hydroxyurea or mimosine can give you a G1/S arrest.

Nocodazole gives you an early mitotic arrest

RO-3306 gives you a G2/M arrest

As someone mentioned above, long treatment with these maybe toxic, so you should optimize, or even use a combination of two inhibitor e.g. thymidine followed by nocodazole (You can find several protocols in the literature). That's the reason why you have to think whether you really need to synchronize. You should tell us more about what you want to do. Are your assays dependent on the cell cycle stage?

The FACS profile using any DNA stain (e.g. propidium iodide, PI) will confirm the cell cycle stage of your culture. You fix 5-10x10^5 cells with EtOH, then stain with PI. In an asynchronous cell culture you should one major peak that comes from G1 cells (X amount of DNA), a second peak from G2/M cells with 2X amount of DNA, and whatever is in between these two peaks are your S-phase cells that have between 1X and 2X amount of total DNA.

Hi Shakti as Thomas suggests a way of ensuring serum starvation brings cells to G0 is to run facs analysis using PI. That way you can at least determine what percentage of cells are in G0. Perhaps certain cells lines will take longer to synchronise.  However as long as you optimise and standardise the length of time of synchronisation for each cell line you will bring the majority of cells to G0

Does growing cells to confluence synchronise cells? Cells that are contact inhibited will arrest growth in G1 or at least have most cells in G1.

If we measure the cell cycle of this population and then reseed the same pool of cells but at lower seeding densities and apply our treatments of interest then measure cell cycle in the treated cells.

Is this considered a cell cycle synchronised experiment?

Hi Badr,

for the first part, I think yes you will most likely at least see a trend towards G1, also depending on you cell line. But then (trypsinizing(?) and) reseeding the cells an then treating them will most likely give them enough time to de-synchronize.

Thanks for your answer Thomas. Would you assume this de-synchronisation be different from flask to flask when the cells have come from the same population of trypsinised cells?

If we measured cell cycle after trypsinsation and before seeding and had a control group grown with normal media and collected cells periodically to monitor cell cycle progression (for example after 6 hours, 24 hours, 48 hours and 72 hours), can we then plot and compare this trend with treatments at 48 hours or 72 hours?

Sorry for dragging on, but I want to try and avoid using any additional chemical agents that may interfere with the study.

I guess the simple answer is: If it was "that easy" that's what people would be doing. But, in the end, you're a scientist - go ahead and try! A small trial experiment should be easy. My guess is it will be murky.

Anyway, why do you want to synchronize cells before drug treatment? Usually it shouldn't be necessary. With a 72h treatment you'll definitely see your effect (like G1 arrest, orso) if there is one. Or is this something more sophisticated?

Thanks very much Thomas. I think you are right, I should carry out a small experiment. I am doing an experiment that is complicated but not sophisticated. A treatment that permits serum starved cells that are mostly G1 arrested to progress into S phase. Serum starvation on its own is damaging enough, I think treatments with other agents will make it even more complicated. I'm new to cell cycle studies and your answer has made me more aware of challenges and possibilities. Thanks for your help.

But wouldn't it then be easiest to seed them in full serum, starve them, treat them (still starved), and then measure. Untreated as control. Ctrl. should then be mainly in G1 as you say and deviations from that would be your drug at work, right? Different time points different concentrations maybe.