Hi researchers,
I'm totally new to cell synchronization experiment. I want to arrest HepG2 cell at M phase and check the expression of a cell cycle related protein. I searched for some prorocols. I saw people use Thymidine arrest and release cells at S phase first then use Nocodazole to arrest cells at M phase.
If my aim is M phase arrest, can I just use Nocodazole?
Also appreciate those who can share an optimized protocol.
Thanks!
Hi,
The problem for harvesting the cells which are arrested in M phase is that the poulation of thoese cells is very low. If you only follow by Nocodazole, you can slightly overcome this problem, however, I highly recommend to use double Thymidine block plus Nocodazole treatment.
To this aim, you have to add Thymidine to your cells on day 0 (preferebly 6 PM). Next day (day 1), you have to wash ur cells twice with PBS and once with media (9 to 10 AM ) and let them grow till 6 PM again. At 6 PM, add Thymidine again till 9 AM of next day (day 3). At 9 AM, again wash your cellstwice with PBS and once with media, and release them to grow for 2 h. Then add Nocodazole to your cells and incubate them foe 10 h. So around 8 PM (harvest point), you will have the cells arrested in M phase.
To increase the efficacy, I recommend seeding the cells in 15 cm dish. At the harvest point, shake your cells at 200 to 240 rpm for 2 mins, keep the media in 50 ml tube. Then add PBS to the dish, again shake for 2 mins and add this PBS to the kept media. Then centrifuge media+pbs at 1000 rpm for 5 mins to pellete the cells. These cells are exactly at M phase.
Most of the remaining attached cells to the dish are also in M phase, but in combination with late G2 cells. Therefore, its better not to use them for your experiment.
Hope it works.
Hi Hossein,
Thank you so much for your detailed protocol! I will try it soon.
Hossein Tabatabaeian , sir, I tried the protocol. After the nocodazole treatment, most of the cells became round and only loosely attached to the substrate. Is there something wrong with my cells? I don't really know what they should look like after the treatment. Thank you in advance for your help!
Mitotic cells are round and have a golden sheen. Nocodazole treatment arrests them in the M phase, so all the cells arrested in the M phase would be round. This is how one can distinguish between the mitotic and non-mitotic cells. They are loosely attached to the culture dish so, I think (by your description) the experiment has worked.
I wash my plates with PBS twice gently and then add a few ml of PBS and tap the plates to dislodge all the M phase cells and then spin down to get the pellet. this pellet can be resuspended in the media and repeated. Be careful not to take out the asynchronous cells (they have a black boundary) while tapping. If you have to just harvest M phase cells you could tap the plate (or spin) in the Noc media and later wash the pellet.
I hope this helps.
Paikwin Li you can reduce Noc concentration. In my hand HeLa is less sensitive than 293T with same dose Noc treatment. Actually M cells are round. Less does may help you to keep them attached :)
Nocodazole arrest cells in G2/M transition, by the inhibition of microtubules formation. I suggest you use the compound RO-3306, CDK1 check-point inhibitor that blocks the G2 to M progression. I have used it and works perfectly, checked by flow cytometry, and a double-thymidine block is not necessary.
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