H2O2 stress induction in RPMI or Balanced Salt? - (Jun/28/2006 )

Hi all,

I'm doing oxidative stress induction and viability assay on my LCLs. I hope that someone in this forum had done this and will share their experience.

My questions are:
1. Can I stress the LCLs with H2O2 in RPMI-1640 medium (without supplements) or must I adhere to stressing cells in Balanced salts such as HBSS or PBS?

2. Do I need to starve the cells prior to stressing it or can I stress it right after seeding it in multiwell plate?

Really do appreciate your replies. Thanks.

Cheers

We had hippocampal neurons, and were told to wait 3-4 days before trying to stress them.

we were told to add H2O2 directly to the medium with different conc. to induce oxidative stress.

I have not tried it though.

Thanks Malik.

I do know that the presence of pyruvate (or pyruvic acid) in the medium is not recommended because studies have shown that it tend to scavenge H2O2 (Desagher et al. 1997). But I'm not sure if the presence of serum will neutralize H2O2.

Has anyone tried stressing their cells in medium containing serum?

Thanks.

Reference:
Desagher et al. 1997. Pyruvate Protects Neurons against Hydrogen Peroxide-Induced Toxicity. J. NeuroScience 17(23): 9060 - 9067

We used Neurobasal for the hippocampal neruons. And serum wasnt added to it but some other additives like ferritin and other supplements. So it wouldnt have mattered here comapred to some other medium, which might contain pyruvate and serum.

Found this , might b interesting for u.

Herpes Simplex Virus Type 1 Vector-Mediated Expression of Nerve Growth Factor Protects Dorsal Root Ganglion Neurons from Peroxide Toxicity

"Hydrogen peroxide treatment of vector-infected primary DRG cultures. Nondissociated primary DRG neuronal cultures isolated from E16 rat embryos were infected at a MOI of 10 with SHN, SLN, or the SN control viruses. Cultures were maintained in growth medium without NGF, except for the mock-infected control, which was supplemented with 100 µg of NGF per ml. At 3 or 14 days p.i., the cells were placed for 30 min in serum-free medium containing 1 mM hydrogen peroxide. After the peroxide treatment, the cells were washed extensively with normal medium, returned to normal medium plus serum for 24 to 72 h, and fixed with 100% cold methanol, and immunofluorescence was performed with either an NGF-specific polyclonal primary antibody (Chemichon) or a neurofilament (NF)-specific monoclonal primary antibody (Boehringer Mannheim) as above. NGF was detected with a biotin-conjugated goat anti-rabbit secondary antibody and extra-avidin-conjugated FITC as described above, while NF was detected with a Cy3-conjugated sheep anti-mouse secondary antibody (1:250; Sigma). Neutral red-stained samples were examined microscopically for the toxic effects of peroxide-induced neurite degeneration by determining the number of processes exceeding 0.25 mm in length."

我也用H2O2诱导氧化应激, 请问要多长时间?

Thanks again Malik. That info was very useful.

I've got translation from a friend and this is what knewman wrote:

" I'm also using H2O2, how long is the incubation"

I am sorry!

you can translate by google language tool.

I'm also using H2O2, how long is the incubation