Question (63684) | HeLa Cell Cycle Lysates: Thymidine-Treated, Nocodazole-Treated & Untreated Asynchronous Control (ab136811)

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Question

Thanks again for your help. I've attached a PDF showing PI histograms of three control samples I collected at different times in my protocol.

I've also attached a paper that I just found with some data that look comparable to what I'm seeing, include a side-by-side comparison of two figures -- one of non-synchronized HeLa cells, and the other of synchronized. Could this sub-G1 hump be a "normal" consequence of synchronizing HeLa cells with nocodazole, then releasing?

For my experiments, I have been synchronizing cells with 2 mM thymidine for 24 hours followed by 50 ng/ml nocodazole for 15 hours, then releasing for 12 hours and collecting 2 hour time points.

Answer

Thank you for your patience.

I checked with the lab and received the following advice:


The sub-G1 population suggests that following release from Nocodazole there is some cell death in the customers experiment. The histograms for the last figure on the ab136811 product page is representative of our results BUT these are taken after Nocodazole treatment (not after release from Nocodazole). Customers treatment conditions of 2mM Thymidine and 50ng/mL Nocodazole are fine for HeLa.

One point of caution for the customer is to be sure that the cells are not overly confluent for these cell cycle experiments… too dense cells will affect cell cycle distribution and health. Their diagram suggests the cells are in the well for >90 hours by the time the treatment scheme is complete so they would have to be seeding the cells pretty thin. I would recommend knocking off the initial 48h incubation post seeding – 8-12h should be sufficient here.




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