I want to check effect of my drug U0126 on cellular proliferation (cell line A498). How can I decide the time of incubation with drug and after how much time I should proceed with proliferation assay?
Dear Chuan..after starvation should I add fbs along with my drug..whats ur opinion??
@Chun-Ta Ho
I would suggest charcoal stripped medium, rather going for FBS medium after starvation.
After serum starvation.You can try using lower concentration of serum along with your drug.Try using 2%FBS.You would probably see a better effect of proliferation...Using higher FBS concentrations could lead to saturation because serum will in itself induce rapid proliferation after starvation.Remember to have a control with 2%FBS in your media.
What a good question. Serum starve can synchronize the cell cycle for sure but how long should you starve the cells will be a realistic problem. I work on pulmonary artery endothelial cell, primary cell culture, and found out the tolerance to serum starve is quite variable. I guess A498 cell lines maybe more resistant to serum starve since it is a cancer cell line. To serum starve for 18 hours (overnight) may give you a better result. I suggest you to mix your compounds, and the control chemical or solvent, into the new culture medium (can be 2-10% FBS) then pour onto the cells. The problem about when to measure the proliferation will rely on which assay you plan to use (MTT, XTT, BrDU, or simply cell count).
Dear Ru-jeng Teng ..I will be performing MTT assay..another question in my mind is proliferation should be studied after the doubling time of desired cell line..The doubling time of my cell line is approx. 48 hours.
Starvation can be for around 4-6h, more of which may not really reflect true capacity ..n about fbs, u usually avoid adding it along with drug as it binds the proteins reducing the availibility also interfering the enzyme/antibody/protein estimations if intended
Hi Deeksha: MTT relies on normal mitochondrial function so basically all live cells with mitochondria function will give you the signal. In this case the doubling time should not be so important. I have done MTT/XTT before and do remember this kind of compund can be added into your culture 4-24 hours before the measurements.
Dear Ru-Jeng Teng..thanks a lot for your reply..While doing experiments lot of questions come to my mind. probably in future I will keep disturbing you..hope you dont mind.
Hii,,Manuel Kaulich ..What should be the approximate time of incubation for PI staining..
Hi diksha , for proliferation try to go for BrdU incorporation as MTT indicated the viability and about the mitochondrial activity but does not give the exact DNA doubling indication. For proliferation assay I would suggest you to seed the cells at low density
( like 5000 to 7000 for per well of 96 well) in serum containing media for overnight. After that you serum starve the cells to synchronize the growth of the cells................For the timing i would advice that while serum starving added around 1% serum or 2% serum as it maintains the health of the cells as well as synchronize the cell growth......for the timing of starvation keep it for 20-24hr is sufficent. After serum starvation you can go for the treatment using your chemical for that you can use very low amount of serum like 0.5% or 1% while giving treatment as this will act as a inducer for proliferation. Give the treatment for the desire time you wish 12h,24h etc and after that add BrdU for 4h or overnight n then proceed for the normal BrdU assay.
Rest consult me for rest. Avoid MTT as indication of proliferation as it does not give the proliferation in real.
For PI staining dilute the PI as the concentration recommended...............10 mins incubation is sufficient .
Most of us believe that starvation for 1-6 hours can synchronize the cell cycle. But this depends on the cell you plan to use. Some primary cell cultures hate serum starve and can not tolerate for more than 2 hours.
Dear Ru-Jeng Teng.. I also work on primary cell culture but have not serum starved the cells but carried on with the proliferation experiment by cell counting. This has led to variable results. Do you suggest that I serum starved the cells? Actually I read that primary cells cannot thrive under serum starvation for long hours.
Do you want to obtain the effects of starvation or your drug on A498 cells ?
You have to determine the time of incubation of cells with the tested agent.
You can analyze the cell count/ cell viability and IC50 value using MTT assay,
I have not come back to this topic for a long time. For the cell proliferation assay, I do believe most researchers will serum starve the cells for a few hours so that all the cells will be in quiescent stage. That may be biological sound and I will recommend. However, I have seen comments from others saying that it is not necessary.
I also had the same questions. I had a toxicological study with HEK293, HEPG2 and NthyOri cells. When I starved the cells, many of the cells could not tolerate the stress and died.
I guess it depends on the type of your study, cell line that you work on and also duration. Alternatively, instead of 0% serum in your media maybe you can try with 2-5% and see if that works.
Ru-Jeng Teng Pro, what if I use reduced-serum (1%) during drug treatment for 24 h for studying cell signaling pathways? I was told by my previous senior mentor that this would synchronize the cell cycle phase (in the quiescent stage). With huge thanks!
If you are studying drug effects on cell cycle or signaling then serum starve should be more productive. I do not know how resilient is your cell line. I have tried simply DMEM with 5% glucose, and the rat endothelial cells survived 24 hours before the treatment. I guess you need to give it a try.
Ru-Jeng Teng Thanks Prof. Mine was human endothelial cells. I use the cells to study drug effects on inflammation and signaling. The cells in DMEM treated with 1%FBS over 24 h seem okay (by visualizing, not cell viability assay), with not much dying but being under stress for sure.
I treated the cells with drugs for the entire 24 h in DMEM with 1%FBS. Do you treat cells by changing back to DMEM with 10% FBS after serum starvation, or kept in DMEM with 1% FBS throughout? Thank you.
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