Hello Nilakshi Patel

I have not worked with H9c2 cells, but I feel you should check for mycoplasma contamination which can affect cell morphology and viability. Routine testing for mycoplasma is crucial for maintaining healthy cell cultures. Also, it is important to maintain proper cell density, and subculture these cells before they reach 80% confluency.

Just for your information, H9c2 cells are known to be sensitive to oxidative stress. They have a high energy demand and rely heavily on mitochondria for ATP production. This makes them susceptible to oxidative stress, as mitochondria are also a major source of reactive oxygen species (ROS). As H9c2 cells are sub cultured, they can undergo changes in their characteristics and become more vulnerable to stressors.

To mitigate oxidative stress in H9c2 cell cultures, you may incorporate the following in your protocol.

1. Maintaining the correct temperature and pH of the culture medium is crucial for cell health and can minimize stress.

2. Avoid excessive shaking or agitation of cells during media changes or transfers, as this can induce oxidative stress.

3. Supplement the culture medium with antioxidants like N-acetylcysteine, vitamin E, or glutathione that can help scavenge free radicals and protect cells from oxidative damage.

4. Be careful of subjecting the cells to hypoxia (low oxygen) conditions, as they can also contribute to oxidative stress. Ensure proper oxygenation and avoid prolonged periods of hypoxia. The oxygen concentration in CO2 incubator should be at around 18-20%.

5. Monitor cell health by checking the oxidative stress markers (e.g., ROS levels, lipid peroxidation) regularly.

Hope these suggestions help!

Best,

Thank you so very much for your response, Malcolm Nobre !