Indirect measures like BrdU labelling require averaging the population behavior. Live cell imaging is a direct approach; are there other direct approaches? And how many cell cycle measurements would be enough?
Hi Bridget
Is your experminent in vitro or in vivo? One method that is gaining popularity at the moment is a dual pulse chase with nucleoside analogues, usually CIdU and IdU. These are both similar to BrdU in that they incorporate into the replicating DNA, however they can be easily distinguished by commercially available antibodies. Generally one would pulse with one label, wait a defined period and then pulse with the second - what you are looking for are cells that are positive for both labels, ie cells that have re-entered the cell cycle. This will give you a minimal cell cycle time for your cell(s) of interest.
We use this method in house to follow mouse mammary stem cells in vivo, however it is also used in our neighbouring labs to look at interfollicular epidermal and hair follicle stem cells, and also to look at Lgr5 and Bmi1 positive stem cells in the gut.
If your experiment is in vitro only then this method is probably not the best, in which case there are many methods you can choose. I am assuming that you might not want to synchronise your populations or to analyse whole poplulations of cells at once, if this is the case then the FUCCI lenti reporters (eg. http://www.mblintl.com/literature/fucci-visualize-cell-cycle) might be of interest to you. With these you can use time lapse fluorescence microscopy and watch each individual cell as it changes from green to red and back to green again as it traverses the cell cycle. You can either buy these from Clontech or other suppliers, or for a more cost effective option you could get them off a colleague or construct your own.
If you choose the FUCCI sensor system and combine it with image recognition software (eg ImageJ or CellProfiler) then you will be able to report on every cell in every well of your culture and under every condition, giving you a limitless sample size.
Hope this helps
Al
Synchronizing cells followed by the measurement of time between two mitotic cycles is a quite good approach. However, this can be applied with high fidelity only for homogenous cell populations like cell cultures.
Hi Bridget
Is your experminent in vitro or in vivo? One method that is gaining popularity at the moment is a dual pulse chase with nucleoside analogues, usually CIdU and IdU. These are both similar to BrdU in that they incorporate into the replicating DNA, however they can be easily distinguished by commercially available antibodies. Generally one would pulse with one label, wait a defined period and then pulse with the second - what you are looking for are cells that are positive for both labels, ie cells that have re-entered the cell cycle. This will give you a minimal cell cycle time for your cell(s) of interest.
We use this method in house to follow mouse mammary stem cells in vivo, however it is also used in our neighbouring labs to look at interfollicular epidermal and hair follicle stem cells, and also to look at Lgr5 and Bmi1 positive stem cells in the gut.
If your experiment is in vitro only then this method is probably not the best, in which case there are many methods you can choose. I am assuming that you might not want to synchronise your populations or to analyse whole poplulations of cells at once, if this is the case then the FUCCI lenti reporters (eg. http://www.mblintl.com/literature/fucci-visualize-cell-cycle) might be of interest to you. With these you can use time lapse fluorescence microscopy and watch each individual cell as it changes from green to red and back to green again as it traverses the cell cycle. You can either buy these from Clontech or other suppliers, or for a more cost effective option you could get them off a colleague or construct your own.
If you choose the FUCCI sensor system and combine it with image recognition software (eg ImageJ or CellProfiler) then you will be able to report on every cell in every well of your culture and under every condition, giving you a limitless sample size.
Hope this helps
Al
Hi Bridget,
I use Flow cytometery to assess cell cycle in vitro cultured cells. For in vivo I tried BrDu and IDu. Later works better.
thanks
RK
Since you ask for direct methods I would say that live cell imaging is the only method you should use. No need for markers; just from cell division to cell division.
Hi Bridget,
If you are working with cultured cells, propidium iodide staining followed by flow cytometry is a good option. But first, you must synchronize your culture in the phase of your interest. There are many methods, depending in which phase you want to synchronize them. If you want to study the complete cell cycle, you can arrest cells in G0 by replacing the culture media for one without serum.
You asked about sample size. Well, for flow cytometry analysis you only will need 10,000 cells (in fact you need more than that, but the cytometer will record only 10,000 events).
Good luck!
Sandra
A problem in cell cycle time measurements is the G0 or non-growth fraction. The tradiational way to avoid their complications of the results is the per cent labeled mitosis technique in which cells are labeled in S phase (e.g., BUdR) and then sequential samples are taken and checked for % labeled mitoses. This eliminates the non-growing cells from the measurement. There are many papers in Radiation Research that have used this method. See also Hall & Giaccia, Radiobiology for the Radiologist, pp 376-388, 7th edn, Lippincott, Williams & Wilkins Philadelphis 2012.
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