I am interested in checking if the cell cycle is accelerated in my cells of interest compared to control (and if possible, calculate cell cycle length). The papers I've looked at so far have used the BrdU EdU method where EdU is given at T0 and BrdU at T0+2hr. Based on the number of BrdU EdU Sox2 triple positive cells the authors concluded whether cell cycle had been accelerated or not. I wonder if this is an accurate method and whether there is a better approach. I say this because, isn't it possible that BrdU and EdU were both incorporated during the same cycle as compared to two different cycles that the authors assume? Any clarification or suggestions would be greatly appreciated.
You have at kest two approaches and I am pretty sure that RG members will propose you a bunch of other ones.
The first approach I have in mind is rather conventional and refers indeed to technique like the one you describe. Here attached is a chapter from a book (Ian Tannock) in which a tritiated thymidine-related test is used.
The second one is quite diferent and relates to the use of computer-assisted phase-contrast microscopy.
Here attached you have some articles describing the potentiality of this approach and also films illustrating the behavior of cancer cells submitted to various experimental conditions.
We routinely perform computer-assisted phase-contrast microscope analyses of living cells during 72h of observation (i.e. at least two dooubling times. The cells (whatever normal or cancerous) are cultured in plastic flasks containing buffered media, but in an incubator without CO2 regulation (while thermoregulated at 37°C).
You can see in the movies here attached the “morphological effects” observed with two very distinct compounds. Each film results from 1,080 digitized images taken every 4 min during 72h and compressed into a 40-60- sec-film. Each film can be seen with softwares freely available from the net. The counter with yellow numbers that you can see in some of the attached films gives you an estimation of the “dynamics” (in terms of number of hours of observation) of the cell behavior.
The attached films illustrate two markedly distinct situations.
The first set of experimental condition refers to human HS683 malignant oligodendroglioma cells (see QVM-1 Le Mercier et al., Neoplasia 2009) left untreated (control; QVM-2 Film Hs683 Control) or treated (QVM-3 Film Hs683-Ferrocifene) with a ferrocifene derivative. These are these morphological observations relating mainly to the enlargement of the treated Hs683 cells as compared to the control ones (without “frank” death processes (see below what we mean as “frank death processes for sphaeropsidine A)) that led us to formulate the hypothesis about potential induction of senescence. The rest of the story is detailed in QVM-4 Bruyere-Mathieu et al. J Inorg Biochem 2014). We estimate that we are facing here cytostatic effects. It is thus possible to calculate from these films the doubling time of individual cells from one mitosis to the next mitosis. It is easier to do that for an experimental condition that "speeds up" the cell cycle than for the reverse situation.
The second set of experimental conditions refers to human SKMEL-28 melanoma cells left untreated (QVM-5 SKMEL-28 Control) or treated with sphaeropsidin A (QVM-6 SKMEL-28 Sphaeropsidine A). These experiments actually helped us to appreciate the cytotoxic effects of sphaeropsidin A (a more “in-depth” analysis is provided in QVM-7 Mathieu et al. CMLS 2015).
Whatever it can be, once a researcher “saw” what happens in the flask containing the cancer cells (when using her / his compound(s) of interest), she / he can thus use other technique, including flow cytometry, to analyze precise cell cycle kinetics or cell death-related parameters. The QVM approach described here also enables the duration of mitoses to be estimated.
Two articles are also here attached with the description of what you can do with this QVM approach.
If the "global growth ratio" as defined in the attached articles) increases in the "treated" conditions as compared to the control one, this means that the cell cycle has shortened. As you will see, you can also evaluate the duration of the mitosis.
Best regards
Robert
I like what Robert has offered. I am not sure what experiments you have already conducted to monitor cell cycle, but if you haven't already done so I would recommend simple FACS analysis of cell cycle using PI or another DNA stain. You can synchronize your cells at G0/G1 using serum starvation, at G1/S with double thymidine block, G2/M using Nocodazole, or metaphase using taxol. You can then release your cells to cycle, collect cells at appropriate time points, stain the DNA, and analyze the number of cells in each cell cycle phase to monitor progression. Another approach is to use fluorescent cell cycle reporters (like Premo™ FUCCI Cell Cycle Sensor (BacMam 2.0)) which express different fluorescent proteins at different phases.
Good Luck
Thank you for your suggestions. I will study them closely and get back to you.
The flow cytometry methods let you label the DNA (PI) and based on the content (2n to 4n) estimate the fraction of cells in G0/G1, S, and G2/M. To get phase durations, you need to know the cycle time (Tc). Using PI intitially and then a flash label with EthBr followed by the Anti-BrU-DNA antibody, one can measure the S phase cells (with their 2n to 4n DNA contents, on a second axis (the 3rd is frequency of cells in that group). The PI staining labels all cells, including those not in cycle, so the Growth fraction becomes a parameter here.
A much better way is the classical % labeled mitoses method. One gives a short duration (flash, 15 min) exposure to 3H-thymdine or to BUdR and then at regular time intervals (15 min, 1 hr) take replicate flasks and add colcemide for 15 min to collect mitotic figures. Initially, there will be no labeled mitotic figures since the label is incorporated only into S phase cells, so cells must progress from the end of S through G2 to reach M and show labeled mitotic figures. The bell-shaped curve starts to rise when the cells at end of S when label was added have reached the beginning of M so this duration is TG2. The curve rises to 100% labeled mitotic figures (only cycling cells have been in S and are now in M) and the straight line slope from the time of beginning of the labeled mitoses to 100% labeled mitoses is TM, the duration of M. When the cells in the beginning of S have finally cycled through M, the % labeled mitoses starts to fall and the time between the 50% points on the bell-shaped curve is TS, the duration of S. The time between the first peak and the second cycle peak is Tc, the cycle time, TG1 is the Tc -(TG2+TS+TM). The precision of this method is excellent and comparing untreated vs drus or radiation treated cells allows one to see where the block is and how long.
A caveat for the drug synchronized cells is that the drugs block a pathway, usually something necessary for DNA synthesis or mitosis, so progression of DNA synthesis or mitosis is stopped, but other cytoplasmic processes may continue, leaving one with a cell population in different parts of the cell cycle in different parts of the cell. The best selection method, applicable only to in vitro monolayer culture cells, is the mitotic shake off method, where mitotic cells are detached from the culture surface by the shear force of the sloshing media against the rounded (higher cross sectional area) mitotic cells, collected and then placed into a new culture flask with fresh media. to grow for various periods of time, providing samples at different stages of the cell cycle for analysis. See Terasima & Tolmach for details of this method. It had also been automated by Talandic.
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