We wanted to test the cleaved caspase3 levels in apoptosis cells which was induced by hydrogen peroxide by using Western blot. However, there always had no results (no bands indeed) for a long time. Do somebody who has experience on these experiments, please give some advices to me. Thanks a lot!
I have no personal experience with these cells but in literature you can find some studies on peroxide-induced apoptosis in RAW264.7.
1. Fong CC1, Zhang Y, Zhang Q, Tzang CH, Fong WF, Wu RS, Yang M. (2007). Dexamethasone protects RAW264.7 macrophages from growth arrest and apoptosis induced by H2O2 through alteration of gene expression patterns and inhibition of nuclear factor-kappa B (NF-kappaB) activity. Toxicology 236(1-2):16-28.
2. Zhang Y1, Fong CC, Wong MS, Tzang CH, Lai WP, Fong WF, Sui SF, Yang M. (2005) Molecular mechanisms of survival and apoptosis in RAW 264.7 macrophages under oxidative stress. Apoptosis 10(3):545-56.
As to t-BHP, there is one report but the authors did not observe apoptosis in contrast to the title
Han L1, Du LB, Kumar A, Jia HY, Liang XJ, Tian Q, Nie GJ, Liu Y. (2012) Inhibitory effects of trolox-encapsulated chitosan nanoparticles on tert-butylhydroperoxide induced RAW264.7 apoptosis.Biomaterials. 33(33):8517-28.
Good luck!
Hi, Yao, have you observed cell death after treatment with H2O2?
How long do you perform treatment ? Perhaps, you can try TUNNEL labelling, this method will shown oyu better results. In addition, I think that better to use t-BHP instead of H2O2.
I used 0.5，1，2mM H2O2 on RAW264.7 cells for 2,4,6,8h. There were not obviously cells death. However when I used 5mM for 4h, the cells were almostly dead. For I used H2O2 as the agent for my anti ROS study, I would like to use the same stimulant in my apoptosis study. But I also want to konw the protocal of the t-BHP in apoptosis study on cells in your lab. I sincerely appreciate for your help !
Yao, I think H2O2 could be a treatment too harsh for inducing apoptosis and it will induce necrosis..A similar phenomenon happens to me, but in a different model (SH-SY5Y). If I use too little H2O2, cells would not die, while just a step up and all die of necrosis. Also keep in mind that H2O2 is quite volatile, thus prolonged treatment are useless...usually 2h is more than enough...I agree with Taras, use t-BHP..and good luck!
I have no personal experience with these cells but in literature you can find some studies on peroxide-induced apoptosis in RAW264.7.
1. Fong CC1, Zhang Y, Zhang Q, Tzang CH, Fong WF, Wu RS, Yang M. (2007). Dexamethasone protects RAW264.7 macrophages from growth arrest and apoptosis induced by H2O2 through alteration of gene expression patterns and inhibition of nuclear factor-kappa B (NF-kappaB) activity. Toxicology 236(1-2):16-28.
2. Zhang Y1, Fong CC, Wong MS, Tzang CH, Lai WP, Fong WF, Sui SF, Yang M. (2005) Molecular mechanisms of survival and apoptosis in RAW 264.7 macrophages under oxidative stress. Apoptosis 10(3):545-56.
As to t-BHP, there is one report but the authors did not observe apoptosis in contrast to the title
Han L1, Du LB, Kumar A, Jia HY, Liang XJ, Tian Q, Nie GJ, Liu Y. (2012) Inhibitory effects of trolox-encapsulated chitosan nanoparticles on tert-butylhydroperoxide induced RAW264.7 apoptosis.Biomaterials. 33(33):8517-28.
Good luck!
Above answers are good. I have not worked with RAW cells, but tert-butylhydroperoxide is usually a better choice. Its best to standardize the dose of tBHP required with MTT assay. Do you want to get IC50 or IC10 value of tBHP ? Its possible that very concnetrationof tBHP (IC 10) will induce enough free radicals to trigger caspase. Higher tBHP concentratiion (IC50) may cause necrosis. So, these dose studies are easily done. Hope this helps!
SOme cells (MCF-7) lack caspase 3--so its worth checking which caspases are expressed by RAW cells.
Another point: Each assays has different issues with sensitivity and specificity-even if controls are good. So, its always good to use 2-3 independent assays for measuring apoptosis. The t-BHP dosage and exposure time must be carefully chosen to avoid necrosis. Good Luck!
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