Hi, I haven't worked with S2R+ cells much, mainly S2 cells. I have yet to be able to synchronize S2 cells at all. Wouldn't be surprised to find that S2R+ cells are similar in that regard. However, you can arrest them during different stages of the cell cycle using various drugs or RNAi of some key genes. First, stop using nocodazole (doesn't work well with fly cells and its not reversible). Instead, switch to colchicine - 30uM final concentration. I treat cells for about 12 hours, no more than that, and can obtain ~20-25% mitotic arrest. Cultured fly cells don't arrest well in mitosis for several reasons: less kinetochores equals a less powerful spindle assembly checkpoint response, cell death, and mitotic slippage -- I believe that all contribute to an overall crappy mitotic arrest. But maybe 20-25% arrest will be sufficient for your experiments. You can contact me for additional protocols if you'd like.

Hi Gregory, thanks for you answer! I'll try with colchicine 30uM for 12 hrs..Have I to harvest cells by "mitotic shake off" (more S2 cells are floating in my hands) or have I to harvest all the cells by scraper?

Hi Gregory !

I am not an expert in insect cells, but I think that pre-synchronization of cells in S phase will increase the efficiency of synchronization in mitosis.

You can see my review of cell synchronization

Rustem Uzbekov

https://www.researchgate.net/publication/8514556_Analysis_of_the_cell_cycle_and_a_method_employing_synchronized_cells_for_study_of_protein_expression_at_various_stages_of_the_cell_cycle?ev=prf_pub

Article Analysis of the Cell Cycle and a Method Employing Synchroniz...

From my experience colchicne will be ok.

Hi Luca,

I have dealt with similar issues of syncing cells but not in a mitosis specific context. I have used S2, S2R+ and primary larval plasmatocytes (ex vivo culture) toward the aim of inducing migration. So far only successful with ex vivo plasmatocytes. The one thing that I have found is the cells required a uniform extra-cellular matrix (ECM), in my cases collagen IV but in most other cases ConA is used, and also a developmental hormone 'kick' as such that was applied during the arrest of the cytoskeleton. The output that I observed was that >60-70% of the plasmatocytes were motile with migration velocities all within the same range.

So with your case of synchronising mitosis within S2R+ cells I would suggest, as an out-of-the-box idea, to ensure a uniform ECM (ConA should serve its purpose and is most commonly used for Drosophila cell culture at 0.05mg/mL) spread across your well bottom (I would simply apply 3-5 mL and leave for greater than 3 hours then remove and leave to air dry over night in sterile conditions). I would then plate my cells, allow them to adhere + spread, then add the colchicine at 30uM as suggested by Gregory, allow for its affect to take place (~2 hours or you could test this for in lab controls) and then add 20-hydroxyecdysone (Ecdysone) at a final concentration of 1uM (for in vitro cultured cells) and then leave to incubate for the remainder up to 12 hours. Once that is done, simply remove the old media and replace with fresh media (with additional ecdysone) for your experiments.

Now the concentration of ecdysone may require optimising as the concentration and time of incubation varies, such as in my case with plasmatocytes incubated for only 3.5 hours with a 30% V/V concentration of ecdysone in ex vivo. For a little help on the background I would suggest this paper:

http://bio.biologists.org/content/early/2013/11/11/bio.20136619.full

I wish you the best of luck with you further experiments, and please let me know if you test out the ecdysone as that would be an interesting result if it works.

All the best,

Chris

you can synchronise S2 cells with ecdysone (nocodazole does not work in S2) and in G1 using hydroxyurea on about 10 days cultures and release. can send precise protocol if needed.

Hi to everyone,

First of all, I would like to thank everybody for your suggestions.Now I know that my cells are "normal"!

I'll try to treat the cells with colchicine 30uM for 12hr at first. Alternatively, I'll use Christopher's protocol.

I'll let you know.

Ps. Mitotic shake off or not?

Cheers,

Luca

Hi Luca,

It is possible to obtain synchronized populations of S2 cells using centrifugal elutriation, which avoids the need for reagents that activate checkpoint responses.

http://www.ncbi.nlm.nih.gov/pubmed/22929773

If you're interested to read more, my former lab studied the topic of cell-cycle synchrony and found for e.g. in S. cerevisiae, even the use of alpha factor, while arresting visible measures of cell-cycle progression such as budding and DNA replication, does not stop the transcription of a majority of cell-cycle regulated transcripts. As a result, they only use centrifugal elutriation as a synchrony method for any transcription-related studies that they do.

Hi Luca, to better answer your question, I need to know what you're trying to do. Do you simply want mitotic cells to do immunostaining or are you trying to collect a mitotic population of cells to make lysates for, say, Western bllots? We tried mitotic shake off with S2 cells and this does not work well because S2 cells do adhere to TC-treated plastic tightly throughout the cell cycle. You can blow them off the surface with easy pipetting. However, S2R+ cells adhere more tight (you can still simply pipet them off the bottom of the flask) but shake-off may work with them. If you plan to do immunostaining on mitotic cells (and you don't care about microtubules of course), then the colchicine treatment will work, 12 hours later resuspend the cells and transfer them onto ConA-coated coverglass and prepare them for immunostaining. You will definitely find all the mitotic cells that you need to image, no need to first shake-off. If however you plan to do something else with the cells, like, prepare lysates for immunoprecipitation, then do not first place them on surfaces coated with ConA because this will cause them to attach so tightly that you kill them trying to get them off. Shake-off in this case may help and you may need to invest some time experimenting with the above synchronization techniques to increase the percentage of cells in mitosis. In our lab, use of drugs such as hydroxyurea and ecdysone work well in arresting S2 cells but we have not been successful in releasing them (I would like to explore this some more though) and after ~3-4 days in the drug, the cells die. Hope this helps.

Oh, sorry, in the above answer, I meant to say that S2 cells DO NOT adhere tight to TC-treated plastic tightly.

Hi Gregory,

Thanks for your answer. I want simply enrich in mitosis because I want to try to make lysates for IPs. To do this, I would like to separate synchronized cells by the asynchronous ones (if I can with mitotic shake-off).

Okay, in my lab, this has been impossible. But other folks here claim that it can be done using the right synchronization procedure. I'd love to be able to get this to work in my lab. I'd also love to see a reference to a paper from someone claiming to have successfully synchronized S2, Kc, or S2R+ cells (in culture and not cemented on ConA) and be able to reproduce it. It may be out there though, just haven't seen it.

Hi Luca,

check this publication:

"Isolation of protein complexes involved in mitosis and cytokinesis from Drosophila cultured cells.

D'Avino PP, Archambault V, Przewloka MR, Zhang W, Laue ED, Glover DM."

They use MG132 treatments for several hours before lysis. Note this is NOT synchronization (I agree with Gregory) but enrichment in the fraction of mitotic cells.

Good luck

Régis

Hi, have ever syncrohonized G1/S in S2 cells?

To do this, we use mimosine 0.5 mM final dissolved in media.

 Hi  Gregory, I am currently try to synchronize S2 cells at G1, S, and G2 phase. How did you make the stock solution for minosine? is the synchronization efficiency good for those stages, rather than M phase? Thank you in advance.

Hi, I believe that I made a 100 mM stock in media at kept it at 4C.  In my hands, an overnight treatment results in ~90% late G1/S-phase arrest.  E2F RNAi also works great at arresting S2 cells in G1-phase.  Arresting cells is no problem, getting them to resume cell cycle progression is the problem.  

that's great, how good for S and G2 phase?(would you mind give the lot number of mimosine?)

We found that nocodazole is not as potent as colchicine when injected into early embryos to induce mitotic arrest.