I am inducing ischemia by OGD in vitro. If I want to estimate mitochondrial membrane potential or look at ROS levels by means of fluorescent dyes, can I fix the cells and then stain for these? I was told that if I fix the cells, the results may not be accurate. But I am worried about staining them at a live state as the cells might be subject to reperfusion when I take them out of the hypoxic chamber.
Also, if I want to check fluorescence by plate reader, can I do so on fixed cells?
Thanks!
Mitochondrial membrane potential is a function of living cells. Fixed cells are dead and therefore do not maintain a membrane potential and do not produce ROS.
If you want to keep the cells alive but hypoxic, can you grow them in some sort of microscope-observable chamber with the atmosphere replaced by inert gas, such as this one?
https://jalinklab.nl/microscopy-under-hypoxic-conditions/
Article Imaging of oxygen and hypoxia in cell and tissue samples
When assessing mitochondrial membrane potential or ROS levels in cells, it is generally recommended to use live cells rather than fixed cells. This is because fixation can alter the cellular physiology and affect the results of the assay. However, if you are concerned about reperfusion injury when taking the cells out of the hypoxic chamber, you could consider using a mitochondrial-specific dye that can be added to the cells before they are placed in the hypoxic chamber. This way, you can assess mitochondrial membrane potential or ROS levels while the cells are still in a live state.
Regarding the use of a plate reader, it depends on the specific dyes and assay conditions you are using. Some dyes and assay conditions may be compatible with fixed cells, while others may not. It is best to consult the literature or product information for the specific dyes you are using to determine if they can be used with fixed cells. It is also possible that you could try a few different dyes and methods to find one that works best for your particular experiment.
In summary, you can fix the cells and stain for mitochondrial membrane potential or ROS levels, but it is recommended to use live cells for this type of assay because fixation can alter the cellular physiology and affect the results of the assay. However, if you are concerned about reperfusion injury when taking the cells out of the hypoxic chamber, you could consider using a mitochondrial-specific dye that can be added to the cells before they are placed in the hypoxic chamber. Whether you can use a plate reader on fixed cells depends on the specific dyes and assay conditions you are using.
Thank you so much Adam B Shapiro , we do not have the facility to have a chamber which can be observed under the microscope, which is why the issue.
Ahmad Al Khraisat thank you for the clarification! However, if I want to give hypoxia/ischemia for several hours, would addition of dye for so many hours make sense?
Yes, adding a dye for several hours during hypoxia/ischemia can make sense, but it depends on the specific dyes and assay conditions you are using. As I mentioned before, it is best to consult the literature or product information for the specific dyes you are using to determine if they can be used with fixed cells and for how long. However, it is generally recommended to use live cells rather than fixed cells when assessing mitochondrial membrane potential or ROS levels in cells, as fixation can alter the cellular physiology and affect the results of the assay. If you are concerned about reperfusion injury when taking the cells out of the hypoxic chamber, you could consider using a mitochondrial-specific dye that can be added to the cells before they are placed in the hypoxic chamber. This way, you can assess mitochondrial membrane potential or ROS levels while the cells are still in a live state.
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