Has anybody here used a single fluorescent marker to reliably score the cell cycle in a variety of cells? The constraints of the experiment are that cells need to grow over 3-5 days without ill effects of the marker and cells will only be imaged at the most once every twelve hours. That means the cell cycle stage of the cells needs to be identifiable without tracking individual cells over the course of a cell division.
I am looking at the Cell Cycle Chromobody (http://www.chromotek.com/products/chromobodies/cell-cycle-chromobodyr/) or a single marker of the Fucci combination (e.g. http://www.lifetechnologies.com/order/catalog/product/P36237). But it seems that either scoring of the cell cycle stages is ambiguous, requires continuous cell tracking, or requires an additional marker.
Does anybody have any experience with this?
Studying cell cycle changes in vivo can be very difficult, I am not sure if this helps, but here is an article that may be of use to you.
http://www.ncbi.nlm.nih.gov/pubmed/15701967
Unless I got it wrong, it is impossible to track cell cycle progression getting data every 12 h. DNA replication and mitosis are much shorter than that. On the other hand, there is not a single marker to discriminate all cell cycle phases. If I were to select a single marker I would use Geminin (S-G2-M) but, again, 12 h-lapse is huge for the cell cycle.
Hello Mario!
You must first thoroughly examine the cell cycle of your cell culture - the average, minimum and maximum durations of each phase of the cell cycle. It is also necessary to determine what percentage of the cells can be in phase G0 at a certain cell density.
Also need to know how much varies (relative to diploid) amount of DNA in cells in G1 phase of the cell cycle - is it aneoploid or polyploid variation for significant part of cells.
You can then 15-30 minutes before fixing cells enter the solution of BrdU what identify after fixation using specific antibodies. (There are more modern visualisation protocoles).
The cells in mitosis can be discerned simply in phase contrast. Cells with BrdU incorporated into the nucleus are in phase S. Cells with small unlabeled nucleus are in G1 phase of cell cycle. Cells with big unlabeled nucleus are in G2 phase of cell cycle.
It is certainly possible to determine DNA content in nucleus of each cell by cell microphotometer .
Perhaps it will be useful for you to read my review about cell cycle analysis and cell synchronization . You can find it on my ResearchGate page.
Best wishes,
Rustem Uzbekov
Ref.
Uzbekov R.E. (2004) Cell cycle analysis and study of dynamics of protein expression level on different phases of cell cycle using synchronized cells. Bichemistry (Moscow) 69 (5), 597-611.
Maybe adding titrated amount of Hoechst or DAPI into your culture medium may provide a way to identify the G1, S, G2/M phase over a long time period, but you first need to be sure that the concentration you add to your culture does not inhibit the normal growth of your various cells type (compare culture without stain with culture with stain over the time course of your experiment and count your different type of cells, if there's no difference then you have your solution.... if not... well... we'll need to find another way of doing it....)
Hope this helps,
Fred
Hi Mario,
Although it is not exactly what you want to do, CFSE cell staining allows you to monitor the number of cell divisions the cells have undergone, using only a single fluorophore. With a time-series experiment this would give complimentary data which can be used to extract information about where cells are in the cell cycle on average. The other parameters fit very well with what you need - 5 day experiment length and measurements every 12 hours. I don't know how well it works with different cell types.
Here is a good reference if you are interested.
http://www.nature.com/nprot/journal/v2/n9/abs/nprot.2007.297.html
Hawkins et al. "Measuring lymphocyte proliferation, survival
and differentiation using CFSE time-series data" Nature Methods 2007.
Cheers,
Liam
- Badges
- Science topic