Inducing apoptosis in Peripheral Mononuclear cells - using Hydrogen peroxide (Aug/07/2007 )
How do you induce apoptosis of Peripheral mononuclear cells using Hydrogen peroxide?
any suggestion and citations will be appreciated
i dont know the answer but i'm interested to know, why hydrogen peroxide?
I may not have complete answer for that Dom. Sorry that I didn't make it clear because I thought it will be a simple question.
I am attempting to use Annexin for Apoptosis assay which is my primary goal. For that, I have to induce apoptosis. I was suggested to irradiate the cells but was thinking of some thing simpler. I came across a few methods to induce apoptosis; but all I need is something that will induce apoptosis and I thought hydrogen peroxide is the by far the simplest.
Do you suggest me any simpler alternative. No body in my lab does an apoptosis assay so there is very less chance that I will find any other inducer. UV Ray can be another alternative but I don't really know how that is done either and don't know how can I have my cells undergo apoptosis rather than necrosis and what are the factors involved.
I will be grateful if you can suggest any simpler method.
Is apoptosis with hydrogen peroxide bad or cause more necrosis than apoptosis?
Thank you for the question Dom
PS congratulations, I read the other post where U said U have become a veteran now. The good thing is, you became veteran by answering the posts and I became veteran by asking more questions - see the difference?
dont have a clue about the hydrogen peroxide (hence the interest) but ive been recomended etoposide as a place to start - i work more with naturally occuring apoptosis and get funny looks when i talk about inducing it in cells (at least for now)
as for questions vs answers - does this mean i know more or just have a bigger ego?
Thank you for the interest and like I said, my intention is to try Annexin V and for that I need to induce apoptosis. I thought it will be a simple thing for those working on apoptosis and will get some replies but seems like Hydrogen peroxide is not much used.
I was not sure if hydrogen peroxide can be used when I started but came across really interesting articles.
I am citing them below for those who may be interested in this.
I will see if I can induce apoptosis in peripheral blood cells with 0.2mM hydrogen peroxide after incubation for may be 4-6h. Then move to FCM analysis with Annexin V-PI.
If you have any suggestion, do write, Dom.
1. A Troyano, et al., “The selection between apoptosis and necrosis is differentially regulated in hydrogen peroxide-treated and glutathione-depleted human promonocytic cells,” Cell death and differentiation 10, no. 8 (August 2003): 889-98. PMID - 12867996
2. A M Gardner, et al., “Apoptotic vs. nonapoptotic cytotoxicity induced by hydrogen peroxide,” Free radical biology & medicine 22, no. 1-2 (1997): 73-83. PMID 8958131
3. Yasuhiro Ogawa, et al., “Mechanism of hydrogen peroxide-induced apoptosis of the human osteosarcoma cell line HS-Os-1,” International journal of molecular medicine 12, no. 4 (October 2003): 459-63. PMID: 12964019
Annexin V decreases the clot rate with decreased lipids. About 10J of UVA light at around 350nm will kill monocytes without lysing them.
I did not get it. I intend to use Hydrogen peroxide to induce apoptosis and Annexin V to stain. Do U suggest me to use UVA instead of Hydrogen peroxide? It will be difficult for me to access UVA.
Help answer my questions on density gradients. Why does the pH change over time?
Thank you Dave2,
I think Hydrogen peroxide will be simpler to start with and will give it a try and if that does not work, will try this. But, I don't have all the materials U have mentioned in my lab and for that I may have to go to another lab.
I am just trying Annexin V; so I will try what is available to me right now. Hydrogen peroxide and then . . dunno. I was suggested Sodium azide but I am not sure if that will work. Azide reduces the metabolism; so in my opinion, it will delay apoptosis instead of inducing apoptosis. Any more opinion?
This is my first post! I'm using H2O2 to induce Apoptosis in HeLa cells along with other cell types which have been transfected with various constructs. The theory behind it is, that H2O2 is a Oxygen free radical source which causes oxidative stress and triggers apoptosis.
If you plan to use it, you first have to do a kill curve to find the LD50 for your cell type (you could probably get it from the literature, but I prefer to figure it out myself!) otherwise you won't be able to distinguish any effect due to treatments etc.
For the nuts and bolts of the experiment, just use the common 30% v/v H2O2 (which btw is 9.7M) and dilute in complete media. BUT make fresh everytime as H2O2 is highly volitile and will degenerate quickly to H20.
To assay for death, just add Propidium Iodide to a final concentration of 5ug/ml right into the well (P.I. is an inter vital dye which only dead cells take up) and you can assay via flow or Confocal. Be sure though, to wash your cells prior to reading.
Hope this helps!