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Paradoxical effects of serum and H2O2 on DCF assay for oxidative stress

DCF

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#1 mfsantillo

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Posted 02 November 2011 - 11:14 AM

Hello,

I am trying to figure out some strange results I have been getting with H2O2, PC12 cells, and DCF assay for oxidative stress. Below are the procedures I have used when treating PC12 cells with H2O2 in media with and without sera.

RPMI+serum procedure:
Load cells with 50 uM DCFDA in HBSS for 30 min, 37 C
Wash with HBSS
Incubate cells with H2O2 dissolved in RPMI+serum
Read fluorescence after 24h
  • Untreated control exhibits 10 pmol DCF per well
  • H2O2 100 uM exhibits 25 pmol DCF per well

RPMI without serum procedure:
Load cells with 5 uM DCFDA in HBSS for 30 min, 37 C
Wash with HBSS
Incubate cells with H2O2 dissolved in RPMI without serum
Read fluorescence after 24h
  • Untreated control exhibits 10 pmol DCF per well
  • H2O2 500 uM exhibits 13 pmol DCF per well
So it seems that the serum is preventing oxidative stress but somehow oxidative stress induced by H2O2 is lower when no serum is present? This seems to be a paradox. Any ideas would be appreciated! Thank you in advance.

#2 zienpiggie

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Posted 02 November 2011 - 11:44 AM

Hi mfsantillo,

I have seen your observation before. The presence of serum does appear to propagate the fluorescence. But just to be clear, why were you using two different concentrations of DCFHDA probe?

#3 mfsantillo

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Posted 02 November 2011 - 11:47 AM

Hi zienpiggie,

Thanks for the reply. I used a higher DCFDA conc in the presence of serum (50 uM) because when I tried to use 5 uM DCFDA in the presence of serum, the signal was too close to background.

#4 zienpiggie

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Posted 02 November 2011 - 11:58 AM

Hi mfsantillo,

I see. It is likely because the serum causes the fluorescence generation in negative control to develop quickly as well. I don't know exactly what is in the serum, but it is possible that there is components in the serum that interact with the probe resulting in the quick generation of the fluorescence for both the negative control or the test compound treated cells.





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