Dear Colleague,

To complete precedent advices, we can consider that ROS production is a natural activity of cell like activated macrophages ie , used by them to kill micro-organisms in lysosomes.

So, activating all cells in vitro like mouse P388D1, human THP1 ... increases ROS production without killing cells, because neutralyzed after their effect inside after of the cell by syperoxyde dismutase.

An frequently, some compounds are used in in vitro experiment either trigering PKC and indirecly production of leukotrienes (ionophore A23187), or directly acting on the plamic membrane (PMA) , or also, as suggested above, LPS reproducing the compounds of bacteria membranes.

The use of the precedent compounds increases greatly indirectly ROS production without killing cells: they can be the result of degradating naturally H2O in hydroxyl radical ie , or ROS produced from the membrane phospholipids or malone dialdehydes.

I hope usefull

Regards

Didier J

Can't you use a mild oxidant that's been shown to induce ROS at sublethal levels? (i.e. curcumin) If you peruse enough papers, you might even be able to build some sort of standard set to compare to.

From my experience I can say that some antioxidants, including catechins, are able to exert pro-oxidant actions without significant killing of cells. You may use these small polyphenolic molecules, ofcourse after dose dependent study.

Hi, Bradley, Yes, you can induce temporary ROS accumulations with low dose of menadione, paraquat, diamide. But in many case this increase will be only temporary, because cells increasing ROS scavenging capacity and try to overcome this dis-balance. Good luck!

Paraquat is good for inducing ROS, but depends on the dose and time. I have found that very low concentration of buthionine sulfoximine would induce ROS generation and kill very less cells (in my case neurons), how ever, you must do a dose response and live dead assay to see the viability.

You can use paraquat, Arsenite, UV and others already announced in the others comments. You can also use proteasome inhibitors. The concentration and time kinetics is very dependent on the cell type. There is only one possibility: try suggested concentrataion and treatment times.

Depending on the cells type you use it is also possible to induce ROS production by Interferons, IFN-beta or -gamma.

Good luck

You may use DMNQ (2,3-dimethoxy-1,4-naphtoquinone) at very low concentrations (0.1 micromolar) or alternatively 2-methyl-1,4-naphthoquinone (Menadione, MEN) at the same low concentration. We have used both in hepatic stellate cells and in bone marrow - derived mesenchymal stem cells. MEN and DMNQ are known to induce in target cells a significant (DCFH-DA detectable) intracellular generation of superoxide and H2O2, respectively (Novo E et al., Journal of Hepatology 2011 vol. 54:964–974)

In our cells toxicity and/or apoptosis were detectable only when using these compounds at concentrations of 10 micromolar or higher. Cautionary note: you should in any case test your cells with a range of concentrations to identify the safe concentration range (i.e. not able to induce cell death) in your specific experimental conditions.

Menadione and paraquat are very good "candidates" be careful with the doses though. It s always good to set up a dose response test to figure out the exact concentration which will give optimal results for you system.

Add thymoquinone 20uM or less .... regine schneider-stock, erlangen university germany

Hi... I have used tert-Butyl hydroperoxide (TBHP) at 50 uM for long treatments (12 or 24h) or at 250 uM for 2hours treatment. However this time and concentration is dependent of the cells, so I recommend you trying different times and concentration for your cell line. Anyway TBHP is better than H2O2 since is lipophilic.

I agree with Fernando, tBHP is a good options as well. But everything is dependent from the aim of your experiments. Do you want to study ROS signalling in cell development? Or cell response? You should distinguish between different ROS types and different ROS localization: menadione, for example, act specifically in mitochondria). tBHP may act on plasma membrane etc. Be very careful with SNP: this compound have a lot of side effects and generally does not induced nitric oxide. At least you have to used iSNP as a control, Good luck!

LPS and Interferon gamma

Most growth factors spike intracelular ROS. It depends what is the end point you are aiming for. very low doses of h2o2 also do the same.

I am agree with Manya, low concentrations of H2O2 (variable according cell lines) can induce ROS without cell death. Another molecule such as COCl2 (an hypoxia mimetic) induces ROS. In all cases, the cytotoxicity must be analyzed in parallel of ROS (DCFH-DA and Hydroethidine).

You may try High glucose concentration or VEGF or free fatty acids, It may work.

Dear Mr. Walters,

by now you have got enough ROS inducers. My suggestion is do not use methods that change the culture condition like high glucose concentration because your aim is to study ROS generation alone. All methods that change the culture condition induce some kind of stress in cells and then the cells produce ROS to its response.

Use any method that do not change the osmolarity or pH significantly. I suggest use very low concentration of H2O2 or other oxidant.

best of luck.

L-Buthionine sulfoximine (BSO) can deplete GSH and induce oxidative stress in cells.

Hi, Bradley, I think now you have enough valuable suggestions. Plesae, consider that majority of the stress- conditions induce ROS accumulations as side effect. So, you need to choose treatments according to your aim, as I mention in previous post. Please, be very carefully with BSO: GSH depletion ultimately will induce ROS accumulations, but also effect is not so fast, and you will observe quite a lot of side effects of the GSH depletion itself, even independent from ROS accumulation. Good luck!

Hi!

Maybe you can try using lipopolysaccharide (LPS).

Good luck! Cheers.

apoptosis is heavily convoluted with metabolic and e.g. hif pathways that are themselves convoluted with ROS, so the best choice will depend entirely and specifically on your hypothesis. pop them in a low o2 incubator and see what happens.

I also suggest to use very low concentration of H2O2 .

Best Regards

A lot of very good suggestions already, I would like to point you into another direction.

Most people think of a bolus addition of "whatever" to increase ROS and this will result in a spike: short, high and in your case obviously deadly. What about tying to induce a small ROS level increase over a longer time? In terms of H2O2 you might think about a slow infusion/infundation instead the one time bolus.

Of course, many of the ROS inducers/sources already mentioned are suitable for this approach as well.

Remember: drastic stresses lead to drastic reactions, physiological reactions are often relatively mild...

I do agree with Taras that the localization of ROS will be important for your experimental setting.

There are several ways inducing extracellular ROS e.g. via Chloramine-T or Lucifer yellow, but these ROS will diffuse through the membrane and already react there heavily.

There are also ways inducing intracellular ROS. e.g. via short-circuiting mitochondria with FCCP or similar agents. Furthermore constant irradiation with intensive light is assumed to create ROS.

If it helps (maybe as a control), you could also try to lower intracellular ROS production, e.g. via Tempol (a chemical superoxide dismutase analogue).

However, you most likely have to check the amount of ROS actually produced, f.i. with a chemiluminescence assay or real ROS sensors.

Dear Colleague,

To complete precedent advices, we can consider that ROS production is a natural activity of cell like activated macrophages ie , used by them to kill micro-organisms in lysosomes.

So, activating all cells in vitro like mouse P388D1, human THP1 ... increases ROS production without killing cells, because neutralyzed after their effect inside after of the cell by syperoxyde dismutase.

An frequently, some compounds are used in in vitro experiment either trigering PKC and indirecly production of leukotrienes (ionophore A23187), or directly acting on the plamic membrane (PMA) , or also, as suggested above, LPS reproducing the compounds of bacteria membranes.

The use of the precedent compounds increases greatly indirectly ROS production without killing cells: they can be the result of degradating naturally H2O in hydroxyl radical ie , or ROS produced from the membrane phospholipids or malone dialdehydes.

I hope usefull

Regards

Didier J

You have to try adding BSO (Buthionine sulfoximine) which deplete the cellular glutathione content and hence you'll offer a great chance to the naturally Produced ROS.

Good luck

I used tBHP, coming with the ROS detection kit. My target cells is IMCD, a kind of kidney epithelial. It works great, Even 4-hour treatment, the cells are happy looking. I can tell more if you like to know.

Thanks to everyone for all of the responses. As many of you have mentioned, I will need to validate whatever I use for my own cells and conditions, so I think I will comb through the extensive list I have been given and try to pick a half dozen or so candidates and then assay them for ROS and for cell death to see what works best. I will post the answer here, if/when I find out. Also, I did not see it here, but I was considering adding HRP (horseradish peroxidase) by itself. Any thoughts on this approach?

Hi, Bradley, HRP is a possible approach as well. But this is dependent from your specific questions. Do you want to study ROS applications itself, or you want to study ROS as consensus of disturbance of cell metabolism. Do not forget to measure ROS accumulations with different fluorescent dyes during your experiments. Good luck!

Hello Bradley!!

Actually there are so many ROS inducers... I like to clear at very first that in case of every ROS inducer, after a certain level (may be called threshold point) every ROS inducers ultimately will cause apoptosis. So all you need to do is to standardize the concentration and time period. Actually it depends upon cell type. You can use rotenone, 6 OHDOPA, MPTP, LPS etc all generate intracellular ROS. H2O2 is an extra cellular ROS. And depending upon your experiment you can estimate the amount of ROS generated by these treatment through different methods. That you can do simply by using DCFH DA method which is very simple and cost effective. Good Luck!!!

We have been using for years UVA-Visible light (320-800 nm). In the alkaline comet assay, the detection limit is 60 KJ/m2 (in primary cultures of human melanocytes)

Also, Hour can use compound called paraquat which was used as herbicide. Such compound is very famous to produce ROS.

hello, i suggest you to use TPA, a phorbol ester derivative. It doesnt kill the cells upto 24 hours but produces Sustained ROS..all the best

Here I would like to point out that ROS is a general term to describe those molecules which are strong oxidization activity to other molecules. Therefore I suggest that you may have to focus on the affect of one or two molecules of ROS to epithelial cells. I think the concentration of H2O2 is around 1 to 100nM and freshly prepared . Good Luck!

Try glutamate. Shall give u a link : doi:10.1371/journal.pone.0039382

Hypoxia or low serum levels can also induce ROS.

Exact Saravana. By the way of HIF 1 alpha pahtway.

See ie the following paper:

"Free Radic Biol Med. 2009 Jul 1;47(1):55-61. doi: 10.1016/j.freeradbiomed.2009.03.027. Epub 2009 Apr 7.

A potential role for reactive oxygen species and the HIF-1alpha-VEGF pathway in hypoxia-induced pulmonary vascular leak.

Irwin DC, McCord JM, Nozik-Grayck E, Beckly G, Foreman B, Sullivan T, White M, T Crossno J Jr, Bailey D, Flores SC, Majka S, Klemm D, van Patot MC.

Abstract

Acute hypoxia causes pulmonary vascular leak and is involved in the pathogenesis of pulmonary edema associated with inflammation, acute altitude exposure, and other critical illnesses. Reactive oxygen species, HIF-1, and VEGF have all been implicated in various hypoxic pathologies, yet the ROS-HIF-1-VEGF pathway in pulmonary vascular leak has not been defined. We hypothesized that the ROS-HIF-1-VEGF pathway has an important role in producing hypoxia-induced pulmonary vascular leak. Human pulmonary artery endothelial cell (HPAEC) monolayers were exposed to either normoxia (21% O(2)) or acute hypoxia (3% O(2)) for 24 h and monolayer permeability and H(2)O(2), nuclear HIF-1alpha, and cytosolic VEGF levels were determined. HPAEC were treated with antioxidant cocktail (AO; ascorbate, glutathione, and alpha-tocopherol), HIF-1 siRNA, or the VEGF soluble binding protein fms-like tyrosine kinase-1 (sFlt-1) to delineate the role of the ROS-HIF-1-VEGF pathway in hypoxia-induced HPAEC leak. Additionally, mice exposed to hypobaric hypoxia (18,000 ft, 10% O(2)) were treated with the same antioxidant to determine if in vitro responses corresponded to in vivo hypoxia stress. Hypoxia increased albumin permeativity, H(2)O(2) production, and nuclear HIF-1alpha and cytosolic VEGF concentration. Treatment with an AO lowered the hypoxia-induced HPAEC monolayer permeability as well as the elevation of HIF-1alpha and VEGF. Treatment of hypoxia-induced HPAEC with either an siRNA designed against HIF-1alpha or the VEGF antagonist sFlt-1 decreased monolayer permeability. Mice treated with AO and exposed to hypobaric hypoxia (18,000 ft, 10% O(2)) had less pulmonary vascular leak than those that were untreated. Our data suggest that hypoxia-induced permeability is due, in part, to the ROS-HIF-1alpha-VEGF pathway."

Best regards

Didier

So I tested a small number of the compounds suggested, and for my cells (renal tubular epithelial cell line), the tert-butyl hydroperoxide (aka Luperox) at low dose (50uM) caused a significant increase in ROS as measured by fluorescence of dihydrorhodamine123 (both numbers of cells and relative optical intensity at 2hrs and 24hrs after treatment), but did not cause increased cell death as measured by uptake of propidium iodide and by total numbers of cells (hoescht) 24hrs after treatment. Thanks to everyone for your helpful insights.

Many if not all disease and stress induce ROS production!!!!!!!!!!!!!

It depends on the sources of ROS, membrane, mitochondria, ER and nuclear, all may induce superoxides, if you want to target certain organelles, you may try specific inhibitors which are commercially available, do a Google search, it will bring most of them.

Hi Brad,

We work with oxidants in many different cell lines and primary cells. To keep your system simple, use H2O2. It will diffuse through water channels of the membrane and hence be in intra- and extracellular equilibrium.

The "killing" by externally added ROS is always a function of the cell concentration (and its size and intrinsic anti-oxidative potential, as well as anti-oxidative enzyme system) used. Further, it is highly dependent on the cell line you are investigating and differs greatly!!!

100uM H2O2 in 1Mio Jurkats per ml medium induces cell death in ~60% after 24h. For THP-1, it is only ~5%. To compare: in primary, non activated T lymphocytes 60uM kills about 75%.

So I would think you'd be safe using 10uM per 1Mio cells without seeing much damage.

A hint concerning you measurement of PI negative cells: cells like HaCaT keratinocytes often simply lyse in oxidative stress so you will not always see the portion of dead cells after 24h. If this is also the case for your cell line watch out for this effect!

Let me know if you have further questions.

Thanks Sander. I had originally tried the H2O2 at low doses but was still getting cell death which was what prompted me to post the question originally. As for using PI as a measure, I also counted total cells (Hoescht and DIC) to hopefully compensate for any loss of PI + nuclei via complete lysis. The reason I wanted the PI was so that I could try to get a measure that was independent of proliferation rate. That being said, the TBHP seemed to work better in my cells since total numbers of cells roughly doubled over 24 hours which was equal to the expansion seen in untreated controls. By contrast, the H2O2 treated samples had decreased numbers of cells after 24 hrs, and some of the other treatments that had been suggested (e.g. BSO) resulted in no significant change in cell numbers at 24 hrs. (though they had increased PI + cells as compared to controls leading me to conclude that they were killing the cells, though they could also have been halting cell division to some extent). If I move to another cell type, however, I will definitely give the H2O2 a try again. Cheers.

I can just propose an idea it may be not practical.

How about promote oxdiative phosphorelation.

Some use galactose media to alter cell metabolism to favor OXPHOS.

What do you think?

Aww. I just realized it was questioned 5 months ago. :P

Please read the article "Morshed SA et al., Journal of AUtoimmunity 2013. you will get all answers. If you have a good fluorescence reader, you can quantify them as well. You can also do live imaging for both qualitative and dynamic time-lapse video microscopy.

Hi Bradley,

hypoxia/reoxygenation process will induce apoptosis. The times of hypoxia and reoxygenation have to be established depending the cell in wich you are working. I am working with epithelial cells too, and 24 hrs of 0.5 O2 hypoxia and 24hrs of reperfusion are perfect to increase the apoptosis incidence.

I hope this is useful for you.

Regards

Jose.

How about ethanol at low concentration? We have observed induction of ROS in EAT cells....

Hy Dr Wlters,

1.- Which kind of cells, cells in primary culture, cellular line, etc.? You need to know the cells.

2.- The role of your cells. Are skin cells, are cancer cells, are neurons, are muscle cell, are stem cells.....?

3.- In which culture camera, do you change oxygen,do you change CO2, do you change temperature, are in hypoxic situations sometimes?

4.- The medium, please. Is it special one with colour, with glucose added, etc?

4.- Do you added antibiotics? Do you added anti-fungus?

When you respond me all the questions we can find some solution.

Thanks

Soraya

@Thimmappa

To my experience with in vitro cytotoxicity test for different companies and different cell cultures, ethanol is toxic for cells in culture, even at low concentrations (simple trypan blue test). So, I suppose it activates apoptosis and generation of ROS.

Regards

Didier

I did my thesis and PhD with alcohol toxicity and I can tell you, if you add alcohol, obviously you are increasing apoptosis and necrosis. So you can not used to mid ROS. Cells with alcohol have toxicity and develop inflammation and oxidative stress. The cells more affected death by necrosis, the cells near the problem death by apoptosis, and many cells near are in memory to destroy quickly when another toxic arrive. So You can do it, better to include another toxic producing oxidative stress without killing yours cells to much. I told you first you need to know yours cells and to know if are ectodermic, mesodermic, etc. Because the sensibility of the cells depend by groups.

Yes, the cell death response to alcohol varies with the cell type. One need to determine acute LC50(ihr exposure) for any compound before using it to induce ROS. Generally concentration at 1/2 to 1/4 of LC50 should be checked for ROS woth out affecting viability.

I was using sulforaphane to induce migration of Nrf2 from cytoplasm to nucleus in murine fibroblasts. This compound elicits such effect via increasing of intracellular ROS generation.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3207051/

You need to fit SFN concentration properly because high concentration also would kill your cells. As I remember I was using SFN at 2,5 uM but I'm not sure. I would verify it.

ROS production increases in stress. If some kind of stress, which the cells could bear, could be introduced in the abiotic form, off course, then the ROS production can be increased. Addition of salts at low concentrations eg. Nacl (~50mM), Alcohols eg ethanol, metal ions. , nanoparticles eg. magnetite nanoparticles, toxins eg. mycotoxin Patulin, UV light exposure etc. Hope these will work.

There are many reports available for the same.

Hypoxia-reoxygenation is not a godd mean, simply because the ROS production is faint, if not null, and this condition is multifactoirial, the cells responding primarily to the decrease in O2. Why not try hyperoxia ? There is an actual production (and transitory) production in ROS, and this induce moderate injury of the cells.

PMA (phorbol 12-myristate 13-acetate) is a good way to produce reproducible amounts of ROS, especially from macrophages and neutrophils.

For reference, you might be interested in Deschacht M., Horemans T., Martinet W., Bult H., Maes L., Cos P. Comparative EPR study of different macrophage types stimulated for superoxide and nitric oxide production. In: Free Radic Res 2010 Jul; 44(7): 763-72

It's a paper produced at our lab.

Cordially,

Jan

You can also use mitochondria toxins, such as rotenone (complex 1 inhibitor), to induce ROS (superoxide) production. Other inhibitors (such as antimycin A, complex III inhibitor) will do the same thing. By carefully controlling the concentration and exposure duration, you are able to see significantly elevated superoxide level, but not inducing apoptosis. There is a good fluorescent dye, dihydroethidium, that can be used to detect changes of superoxide production. This is mitochondria ROS induction.

The agents mentioned by other researchers, such as PMA (a PKC activator) can also stimulate superoxide production via activation of NOXs (mostly NOX2), representing cytosolic induction. Whereas LPS-induced ROS (I believe also mostly superoxide) is generated via activation of TLR-4 receptor.

If you use neuronal cells (such as primary hippocampal neurons), you can use low dose NMDA (a NMDA type of glutamate receptor agonist) to stimulate superoxide production, but not trigger excitotoxicity (high dose of NMDA will).

Low dose H2O2 will do the trick but it depends on the type of cell; need to check LDH release to make sure the employed dose is harmless; a short heat-shock plus nm levels of H2O2 may work. A low dose CCl4 will also do but CCl4 reacts with plastic wares (caution!!); can be induced in sterile glass tubes (within minutes guaranteed) and then transfer into plastic wares promptly.....a lot of work but worth trying and rewarding as long as hypothesis-testing is done right!!!!

Stress and disease induce (re)active oxygen species production!

How about trying low dose of acetaminophen?

Why acetaminophen? Can low dose of any drug induce cellular ROS?? Perhaps it depends on the cell type. Renal or liver cells may get stressed with almost any drug (incl acetaminophen).

Use a low concentration of menadione in your cell medium (check Shalini 2012) for exact concentrations

In my experience Acetaminophen will far less toxic than menadione. In fact acetaminophen is barely toxic in vitro (liver or kidney cells) but highly toxic in vivo. Yes, low dose of any drug can slightly potentiate base-line ROS production; however, the investigator needs to make sure from the lietrature whether the cell line in question has the right kind of CYP450 isozyme or not....I believe, a small manipulation with acetaminophen may work better that menadione or any other compound...

Agree about acetaminophen; which is no more used in France since several years. Effects at the level of entire organism can be different than on cell culture; even if cell cultures are often used during pre-clinical phase of drug development (cytotoxic effect).

In vivo, often drugs can have a toxicity on organs with an elimination or detoxification role (liver and kidney), and these toxicities are the main limitating factor during drug commercialisation.

Acetaminophen was responsible of severe bone marrow aplasia. That could be linked to effect during BM cell line differentiation, more than a direct toxic effect.

Regards

Didier

Agree but disagee that it is discontinued from human use. In Europe it is called paracetamol and it is one of the most popular analgesic known to human todate. It is the most popular OTC product used as a pain killer in the US. Thanks for your note though!! very insightful!!

The contributions above illustrate the characteristic paradigm of of research on ROS.... What are you seeking for? Reproducing natural ways of ROS production or meaningless pharmacological stress ?

Sorry Sidhartha.

I probably made a mistake, because of different international denomination of drugs, or commercial names, with amidopyrine.

So, if acetaminophen is paracetamol, toxicity is not the ones I described, but really an hepatic toxicity. But cellular effects can vary , because its toxicity is dose-dependant (not surpass 4 g/day).

Regards

Didier

No problem Didier....understood.

Pierre---looks like the originator of this question is looking for meaningless pharmacological stress not necessarily to look for pharmacological or toxicological effects; the author of this question is interested in inducing apoptosis unless I misunderstood!

I work in human kidney ephitelial cells (HK-2), here induced oxidative stress with H2O2 200uM without apoptosis until afer 6-8 hours, being that exist different mechanism of cytoprotection such as heat shock protein and Nrf2-Keap1/ARE pathways and others

Please check my recent paper where I have used multiple ROS activators or inhibitors in rat thyrocyte culture. Morshed SA, et al J Autoimmunity 2013.

use of low dos of any toxin or toxicant can induce ROS in your cells (paraquate, organophosohorus and .....)

Xanthine oxidase makes ROS. Complication is that makes Uric acid, a conditional pro- and antioxidant.

The influence of hydrogen peroxide, superoxide anion radical or nitric oxide on cell cultures is highly concentration dependent. The action may result in proliferation or apoptosis or complete cell lysis.

So the use of any ROS /RNS generating compound has to be established experimentally for specific cell line and media.

Try the concentrations of superoxide radical in nano-molar range.

There are various methods that one could use to model oxidative stress in cell culture. Attached is an article that may be of use to you.

http://www.ncbi.nlm.nih.gov/pubmed/1383781

hi Bradley

in my some experiments i tried to test the role of ROS in cell morphology by infection the  particular stable cell line with recombinanat adenovirus expressing Nox1 gene ( NADPH oxigenase ) that increases the ROS, so we can test the the optimal amount of virus the gives a good result.  

Hyperoxia induces a partial depression of cultured cell functions correlated with  free radical production

hypoxia chamber can be usefull, without adding any chemical compounds you can create hypoxia conditions. 

Crucial question, how long do you need to keep your cells alive/viable in the presence of increased ROS production? We have used different types of cells and stimuli, but after 72 h stimulus viability is often affected.

Hi, I don't know what is your intention of studying ROS production. But, I would like to remind you one thing that I have read and experienced. Higher amount of ROS kills cells, Medium or low say 5uM-10 uM ROS (H2o2) shows kind of phenotype changes like say little invasive phenotype but doesn't kill the cells upto 24 h and If you use really low it possibly doesn't show significant effect. Just maintains the growth/survival of cells. You can use TPA (1 or 10 ng/ml), a phorbol ester derivative to induce ROS production. Some cytokines also increase ROS but depending upon cells. There are various sources of ROS (mitochondria, NOX, LOX, XA etc). Make sure about the phenotype and your intention for inducing ROS production.

As said before immune signaling pathways induce ROS and RNA in many cell types. Most cells express interferon receptors and many cell types express TLR4 for LPS signaling. You can also use proteasome inhibitors at low dose applications, but this depends strictly on the cell type and the kinetics of time Inhibitors of the repertory chain will work as ROS inducers as well.

Article Cell culture models for oxidative stress: Superoxide and hyd...

Might be worth reviewing to see if this can give you other ideas.