Chloe Sun

Dear, H2DCFDA can be used for fluorescence microscopy observation of adherent cells.

H2DCFDA is a lipid-soluble probe that can freely pass through the cell membrane and enter the cell. It is hydrolyzed into DCFH (non-fluorescent) by esterase in the cell. When encountering ROS (such as H₂O₂, peroxide, etc.), DCFH is oxidized to DCF (2',7'-dichlorofluorescein) with strong fluorescence, and its fluorescence signal can be observed by fluorescence microscopy (Ex/Em=488/525 nm). The culture environment of adherent cells (such as culture dishes and slides) is suitable for direct observation under a fluorescence microscope, and can clearly present the distribution of ROS in the cell (note: fluorescence is mainly located in the cytoplasm).

Q: How long after staining can it be observed?

A: It is recommended to observe within 30 minutes to 1 hour after staining, and no longer than 2 hours. Pay attention to light protection and washing steps during operation to obtain a more stable fluorescence signal.

The answer to this question comes from MedChemExpress (MCE) Technical Support.

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Hello Chloe Sun

You may follow the protocol provided below.

1. Culture adherent cells on a suitable growth substrate and avoid culture media with phenol red.

2. Dissolve H2DCFDA in DMSO to create a 10mM stock solution.

3. Dilute your H2DCFDA stock to a working concentration, (20µM has worked well for me) in culture media. You may optimize the working concentration in the range 5-25µM. Please note that the working solution must be prepared fresh, and avoid storing or reusing it.

4. Aspirate the culture medium from the cells and wash the cells with 1XPBS.

5. Add the H2DCFDA working solution to cells, ensuring it covers the cells completely.

6. Incubate the cells in the dark at 37°C (or the optimal temperature for your cells) for the appropriate loading time, typically 30-45 minutes.

7. Wash the cells with 1X PBS to remove excess dye.

If you plan to treat the cells, follow the steps below.

7a. Aspirate the H2DCFDA working solution.

7b. Wash the cells twice with 1X PBS to remove any excess H2DCFDA.

7c. If you're studying the effect of compounds on ROS production, you can now add fresh media containing your desired drug or treatment.

7d. Incubate the cells for the required time according to your experimental design.

8. Use a fluorescence microscope with a filter set appropriate for fluorescein (FITC) or a similar dye (excitation/emission around 488/525 nm).

9. Observe and image the cells within 30 minutes to 1 hour after staining, and no longer than 2 hours, for a more stable fluorescence signal.

Important points to be noted.

1. H2DCFDA is light-sensitive, so perform all steps in the dark and protect your cells from light after staining.

2. Avoid phenol red in culture media as it can interfere with fluorescence.

3. Optimize your cell density to avoid too low or too high a signal.

4. Image the cells using low light intensity to prevent photobleaching of the probe.

5. Be mindful of potential artifacts, such as self-oxidation or photo-oxidation.

Include cell-free controls to ensure that observed fluorescence is due to cellular ROS and not chemical reactions with the probe itself.

7. Include positive controls (e.g., cells treated with a known ROS-inducing agent) to validate the staining procedure. Include unstained cells as a negative control.

Best,