I need to detect the ROS levels of the differentiated neuronal cells using DCFDA reagent from Thermofisher. Following are the steps followed:
1. Differentiated neuronal cells seeded in 96-well plate (Black with clear bottom). Incubate for 24 hrs.
2. Treated the cells with H2O2 for 24 hrs.
3. Discard the media and added pre-warmed PBS/HEPES/HBSS.
4. Removed Buffer and stained the cells by adding diluted DCFDA Solution at the final working conc of 1-10 μM.
5. Incubate cells with the diluted DCFDA Solution for 45 minutes at 37°C in the dark.
6. Remove DCFDA Solution. Add PBS/HBSS/HEPES and measure fluorescence immediately to get a baseline fluorescence intensity.
7. If performing toxicity assays, remove Buffer/PBS and add diluted compound(s) of interest. Treat cells for desired time (1 – 6 hours). Include appropriate controls. DO NOT wash after treatment.
8. Measure plate on a fluorescence plate reader at Ex/Em = 485/535 nm (FITC range) in end point mode in the presence of compounds, media, or buffer.
Could someone advise if the protocol is correct as I see a huge signal from the well not treated with any H2O2 (No treatment what so ever).
Mam protocol is correct but if you are getting strOng intensity even in non treated sample then u should diluted DCF more.
Hi Aruna Raja did you manage to correct the problem with your protocol? I am getting similar problem (my untreated sample is giving more signal than the treatment!) Would you please share how you resolved your problem? Thanks
We stain SH-SY5Y cells with 10 μM DCFH2-DA for 30 minutes at 37 °C in the dark. We have no problem with the fluorescent signal.
Could you explain some points? How long do you incubate the cells stained with DCFH2DA before the measurements?
According to point #6, you record baseline fluorescence immediately after washing of the cells. Is the baseline signal high in non-treated wells?
I have some thoughts about this problem:
1. The rate of ROS generation in SH-SY5Y cells is relatively high, and 6-hours incubation is too long for these purposes. So, DCFH2DA can be fully oxidized after long incubation (more than 2 hours) of the stained cells.
2. Check the excitation and emission filters in a plate reader. We had a problem (high signalin all wells) when the emission filter was mistakenly placed in the wrong position.
3. The fluorescent signal is sensitive to the z-position in some cases. Try to calibrate the z-position and choose the optimal value if the software of your reader allows to do it. We calibrate z-position before each measurement. For these purposes, we estimate the ratio signal/z-position for the control well (cells stained with DCFH2) and "blank" well (cells without the probe). What is the signal intensity in a "blank" well?
4. If the signal is very high in a "blank" well, it is better to decrease gain.
If you have an opportunity, check the DCFH2-stained cells under a fluorescent microscope. Use low exposition time for microscopy because DCFH2 is very sensitive to the light, and it can be fully oxidized during 10-30 seconds of continuous irradiation.
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