Is there any way to visualize intracellular ROS production in drug treated MCF-7 cells by using confocal microscopy?, If yes, How?

Hello Swapnaja Gulawani

Yes, you may follow the following protocol.

1. Grow adherent cells in standard cell culture media to an appropriate density on a suitable substrate for live cell imaging (chamber slide, 96 well plate, etc., depending on available microscopy setup). Avoid using phenol red (background fluorescence).

2. Treat the cells with the drug at the indicated concentration for an appropriate time.

3. Wash the cells 1 or 2 times with 1X buffer.

4. Stain the cells by adding diluted DCFDA solution (20 µM DCFDA).

5. Incubate the cells with the above diluted DCFDA solution for 30 minutes at 37°C in the dark.

6. Wash the cells 1 or 2 times with 1X Buffer.

7. Perform live cell microscopy with filter set appropriate for fluorescein (FITC). Maintain low light conditions to avoid photobleaching and photo oxidation of DCF.

For fluorescent microscopy measurement you could visually score cells for brightness and compare between control and samples or use image analysis methods to compare signals on digital photographs of cells.

I am also attaching two papers for your reference.

Article Production and Detection of Reactive Oxygen Species (ROS) in Cancers

https://pubmed.ncbi.nlm.nih.gov/23178299/z

Best.

Taufeeque Ali

I agree with Malcolm. DCFDA - Cellular ROS Assay Kit from abcam (ab113851) uses the cell permeant reagent 2’,7’ –dichlorofluorescin diacetate to quantitatively assess reactive oxygen species in live cell samples.

It measures hydroxyl, peroxyl and other reactive oxygen species (ROS) activity within the cell.

DCFDA is also available as free molecules as ab145286 (Carboxy-DCFDA N-succinimidyl ester).

Kajanthan Piraisoody

You can go for CellROX as previously mentioned or MitoSOX Red (Invitrogen, ThermoScientific). It is depending on which kind of ROS you want to measure. MitoSOX is more specific, measuring the superoxide production in mitochondria.

Ganesh Panditrao Lahane

Yes, we can detect intracellular ROS by DCFDA and DHE staining.

For further details, go through following article.Article Nesfatin-1 peptide protects rat renal epithelial cells again...

Saif Wahid

It is possible to visualize intracellular reactive oxygen species (ROS) production in drug-treated MCF-7 cells using confocal microscopy. Here is a general protocol to achieve this:

Cell Preparation: Prepare a culture of MCF-7 cells and treat them with the drug of interest. Ensure appropriate drug concentrations and treatment durations based on your experimental requirements.

ROS Probe Staining: Select a suitable fluorescent ROS probe, such as 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), dihydroethidium (DHE), or CellROX dyes. These probes are designed to be oxidized by ROS and generate a fluorescent signal. Follow the manufacturer's instructions to stain the drug-treated cells with the ROS probe. Typically, this involves incubating the cells with the probe for a specified time at optimal conditions.

Cell Fixation: After staining, carefully remove the staining solution and fix the cells using a suitable fixative, such as 4% paraformaldehyde. Fixation helps preserve the cellular morphology and prevents probe leakage.

Washing: Wash the fixed cells with an appropriate buffer, such as phosphate-buffered saline (PBS), to remove any residual fixative and staining solution.

Mounting: Mount the cells onto a glass slide using a mounting medium that contains an antifade agent to preserve the fluorescence signal. Ensure that the mounting medium is compatible with confocal microscopy.

Confocal Microscopy Imaging: Place the slide on the stage of the confocal microscope and select the appropriate excitation wavelength for the ROS probe you used. Set the laser power, gain, and other imaging parameters based on the specific requirements of your probe and microscope system.

Image Acquisition: Capture confocal images of the drug-treated MCF-7 cells using appropriate fluorescence channels. The ROS probe should exhibit a fluorescent signal in regions where intracellular ROS production has occurred. Adjust imaging settings, such as focal plane, z-stack depth, and image resolution, to capture the desired cellular details.

Image Analysis: Process and analyze the acquired images using suitable image analysis software. Quantify the fluorescence intensity in regions of interest (ROI) to assess intracellular ROS levels. Comparisons can be made between drug-treated and control cells or between different treatment conditions.

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