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I have followed the modified protocol that was given to me by my senior (who was using for his RAW 264.7 cells - Reference article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308605/ ) for assessing ROS in N9 cells. But, I am not able to standardize it in our lab ... every time there are variations in the results.

Protocol

  1. Seeded 10,000 cells per well in a 96 well plate.
  2. After 12 Hrs of growth, the spent medium is discarded and the cells were washed twice with PBS.
  3. After the PBS wash, 50uM DCFDA was prepared in PBS and 100ul/well was added in 96 well plate and then incubated for 30 min at 37oC and 5% $CO_2$.
  4. The DCFDA was removed from the wells, immediately and cells were washed twice with PBS.
  5. After the wash, to standardize the protocol, the cells were treated with 10uM, 30uM, 50uM ${H}_{2}{O}_{2}$ as a positive control and 1mM and 5mM N-Acetyl Cysteine was used as negative control.
  6. And a set of cells not treated with any compound (DCFDA/${H}_{2}{O}_{2}$/N-acetyl cysteine) are also used as control.
  7. After treating with the compounds, the fluorescence was recorded in multi-plate reader at (excitation -485 nm and emission-530 nm) different time points with a 10 min interval - T0, T10, T20, T30, T40, T50, and T60.

We could not see in difference in different concentrations of ${H}_{2}{O}_{2}$ (newly purchased) and also with N-acetyl cysteine. We would expect to see some difference.

It will be a great help if you can suggest me where I might be going wrong with the protocol or is it anything to do with the seeding density of the cells.

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  • $\begingroup$ Hi and welcome to Bio.SE! This is a good example of a lab-technique question as far as I can tell and I hope someone can point you to the right direction! I've made some formatting edits, but feel free to roll back. $\endgroup$ – James Nov 25 '16 at 5:49

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