I am doing an experiment to investigate the effect of our compound on cell cycle. I will be using Nocodazole to synchronize my A549 cells but see a wide variety of doses when searching the internet, though I most commonly see 50-100ng/mL for 12-16 hours. Most of what I see is for HeLa cells, so I'm guessing it shouldn't be too different for A549s? How long after removing nocodazole should I wait to treat with compound?
Also, as I am new to synchronizing cells, which cells do I use for my experiment? The cells that are floating or the cells that are still attached?
Thanks!
Hello Lindsey.
Not all the cells are not synchronized specially in cancerous cells having varying number of chromsomes due to their cell activities. It would be satisfactory to get higher proportion of synchronization. I would use 18h incubation at a final concentration of 100ng nocodazole. It is critical to select active state of cells cultured at about 50% confluency. Therefore, start with 25% of cell confluency, followed by culturing for 22h. Change the medium containing 100ng nocodazole/ml. Important to make 5000~20,000X stock solution of nocodazole in DMSO, aliquot them in E-tubes, store at -70C until use covered with aluminum foil all the time. At the end of the drug incubation, carefully change the medium without removing metaphase cells. Dissociate with 1/5 dilution of trypsin-EDTA, collect in tube, and wash the remaining drug and DMSO completely. You could use the cells or replate cells depending on your purpose.
Regards.
You shall find interesting protocols in
Zieve G and McIntosh JR. Experimental Cell Research 1980;126(2):397-405...
hi starting with a new cell line, i would do a dose study ..if you are interested to release the cells from the prometaphase block, it is wise to use the minimal dose .this way you will "help" the release..if you use to uch drug, you might create microtubules anomalies which might get you into trouble. prometaphase arrested cells still stick to the culture plate to some extend, so do a mitotic shake off (remove the culturemedia while flushing the media with the pipetor, collect this SN, add PBS repeat the flsuhing/skaking, combine booth) , centrifuge, wash the cell pellets twice with pbs and reseed in a new plate or coverslip polylysine treated if you want to monitor the meta-telophase transition)
if you are only interested to arrest the cell in prometaphase ...100ng/ml should be fine
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