Hello Devi,

how long was pulse in figure 1? Have you used flow cytometry for analysis?

It is definitely something went wrong, because flow cytometry reflected relative duration of cell cycle phase.

In Your case there is almost not G2 ea. cells were arrested.

Please, provide more details.

Pulse in Fig1 was for 2h before harvesting the cells. Yes, analysis was by flow cytometry. CFSE analysis showed that the cells were cycling. There are usually less cells in S and G2 phases compared to G1. The kind of profile I got is what I see in many sources online too. My question is why there are hardly any cells (in late S phase) that take up moderate levels of EdU and have double the DNA content.

Thanks, I agree that G2 cells are less as G1, but not so much less. G2 must be appr 20% but not 0,32%,

For your question, do not forget that you have high thymidine pool inside cells and in short pulse thymidine also incorporated . The most importnat is the ration between endogenous thymidne and EdU what you add.

At early time point you have a lot of endogenous thymidine pool, that's why EdU incorporation (to compare with thymidine) is relatively low.

My best wishes!

Yes, I too was just thinking of a similar explanation. Thank you for the answer!

Very good, many people simple ignore presence of endogenous thymidine pool and its changes through time.

Moreover, it will be nice if you can perfomed determination of G2 duration by measure of the time at which first G1 EdU positive cells were appeared.

It will also give you a prove about cell cycle kinetics.

I hope it will help.

My best wishes!

Yes, that would be interesting to find out! Fig3 is the profile if I pulsed for 24h and Fig4 if I pulsed for 48h. However, as you can see, G2 is still below 1%.

Another question I had was: Say I want to pulse for 48h. Would there be a difference if I added 10uM EdU for the first 24h and then again a fresh stock of 10uM EdU for the next 24h, as compared to letting the first 10uM of EdU stay in the medium for 48h? Also, are there any toxic effects on the cells with such long pulses?

There is a quite good paper about EdU toxicity in 2017. Pereira et al., 2017

After 5-6 h you must have EdU-positive G1 (cells what passed G2-and M).

May be in your case it require longer time, 7-8 hours, but not longer. In the case if you do not have EdU-positive G1, it is a problem with your cells cycle progression, because G2 is 2-3 times shorter as G1 and you should have in cycling cells appr 60%G1-10% S and 30% G2.

Have a nice weekend!

Thanks, I'll check that out. You too!

Pereira, P. D., Serra-Caetano, A., Cabrita, M., Bekman, E., Braga, J., Rino, J., ... & Ferreira, J. A. (2017). Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescence-intensity analysis. Oncotarget, 8(25), 40514.

Article Quantification of cell cycle kinetics by EdU (5-ethynyl-2'-d...

Thank you for the link. I had read this paper briefly and noted that 10uM does not cause much damage. However, the paper shows studies upto 11h and then 5 days of pulse. In the last few days I tried it out myself and found that 24h or more pulse substantially affected cell viability and proliferation. I had tested only 2-3h and 24h, nothing in between. I shall try a few points in between and perhaps stick to less than 12h.

My main purpose of attempting long pulses was to identify slow-cycling cells. With 2-3h pulses, it is less likely that I catch a representative number of cells in the S phase. However, longer pulses are killing the cells. I have seen papers where they give several days of pulse in vivo; the bioavailability might be different, though.

Would you think EdU's harmful effects would be only on rapidly-dividing cells and not on resting cells?

Thanks, You can try to reduce EdU to 5 µM or even 3-4 µM. It will be sufficirntly to get your aim.

Moreover, I would suggest you to ficus on determination of EdU-positîve G1 (ea. G2 duration), But it is alse dependent form your cells type/cell cycle kinetcs. From your data I can conclude that your cells rather arrested in G1/early S-phase because main stage in current moment are G1.

For the rapidly cycling cells you must have significant portions of G2 as well (at least 20%).

I hope this will help.

have a nice day!

Dear Devi, have you got any progress with labelling? Have you tried lower EdU? Yes, EdU is toxic only after incorporation to DNA because have some effect on transposition. There is EdU-derivatives without side effect, and this compound is not toxic.

You can try to order it for your labelling.

Have a nice day!

Thanks for the follow-up. I'm yet to test lower concentration/time. I'll also test if the effect is due to DMSO, though the final concentration is pretty less (0.1%). I shall check out the alternative label too.

Will keep you posted.

Article DNA Damage Signaling, Impairment of Cell Cycle Progression, ...

Please. order EdU derivative which is not toxic. There are sevreal complany what produce it.

Have a nice day!

Article Prospects and Limitations of "Click-Chemistry"-Based DNA Lab...

Dear Devi, how are you, have you got any progress last month?

Plesae, have a look here, but they used mainly 20 µM EdU and confused DNA staiability with DNA contents.

My best wishes!

DNA Damage Signaling, Impairment of Cell Cycle Progression, and Apoptosis Triggered by 5-Ethynyl-20-deoxyuridine Incorporated into DNA

Hong Zhao,1 H. Dorota Halicka,1 Jiangwei Li,1 Ewa Biela,2 Krzysztof Berniak,2 Jurek Dobrucki,2 Zbigniew Darzynkiewicz1*

Thank you for the follow-up. I had gone through the paper you mentioned.

I tried various concentrations of EdU for different durations. If I need to just pulse cells and harvest them, even a 24h pulse seems okay. However, I do not know if the pulse has caused any damage to the cells. On the other hand, if I need to pulse and chase the cells, even a concentration as low as 1uM hampers cell viability and proliferation, if pulsed for 24h. Most cells die of toxicity by 24h of the chase (after the 24h pulse). Essentially, the effect is both dose- and time-dependent; I have tried 1, 2.5, 5 and 10uM for 3, 6, 12 and 24h of pulse. So, no matter the concentration, a 24h pulse is not usable for chase. For shorter pulses, each specific experiment should tell us how much of damage is acceptable. However, the concern is that the method to measure proliferation itself affects proliferation. For now, I shall use sub-lethal conditions as I cannot purchase alternatives.

Thanks! How do you add EdU? with new medium of EdU only? what is the solvent?

If the concnetartion of EdU does not have effect, may be the problem with the solvent? If you use DMSO, concnetartion higher 1 µl/ml is very toxic.

Other solvent may alos have effect.

Try to avoid to addition EdU with chnages of the medium, it is alos have a very negative effcet.

My best wishes!

I add EdU in DMSO. I maintained the amount of DMSO constant in all treatments. I did verify the effect of DMSO (1 µl/ml ) and the cells do as good as the untreated group, with no visible effect at all. I add EdU into the existing medium and mix by pipetting. After the incubation, I wash the cells once and re-seed with fresh media.

Very good protocols! It seems EdU is quite toxic to your cell types, what os not so common effect. In my case even 10 µM EdU is not very toxic, as well in other publications you were read. May be you can try another assay: http://www.bio-rad.com/de-de/product/cell-proliferation-assays?ID=MTFV5T4VY ?

Or EdU for maximal 4-5 houirs after which you can detect EdU-positive G1?

Have a nice weekend!

Thank you for the information. I am already using CFSE in addition to EdU. Though both give me readouts on proliferation, the information obtained from either of them are slightly different. Thinking aloud, for example, even before CFSE peaks start diluting, I see EdU positive cells. While EdU only gives me the cells in S phase, CFSE also tells how many divisions a population has undergone.

Coming back to EdU, I shall perhaps stick to lesser pulse durations for pulse-chase experiments.

Thanks! I hope you washed with conditioned medium after bEdU label.

Moreover, EdU show you cell in S-phase only during forst 20-40 min after addition.

For longer incubation time, even if you washed form EdU, they show cell which pass DNA replication, but not necessary in S-phase.

Have a nice day!

True, I agree. However, I have not observed EdU+PI- cells by a 2h pulse. Maybe the differences in rates of incorporation. Thank you for your suggestions!

By the way, to avoid EdU toxic effcet you can easy add 20% EdU and 80% thimidine insted of 100% EdU.

I hope this may help

ara-EdU

If possible, try to use afr-EDU. It will help for sure!

Thank you for the paper. Looks like the toxicity is greatly reduced. However, I am not sure if I will be able to purchase any more reagents in this regard. If I can, I'll surely go for ara-EdU.