Hello, The two different types of cell death morphology you encountered while culturing bEnd3 cells may stem from several possible reasons. Here are some thoughts:

Apoptosis and Necrosis: The typical cell death observed in the first image is likely apoptosis, which is a form of programmed cell death. In this case, cells break down in an organized manner and do not harm their surroundings. The morphology seen in the second image is probably necrotic death; in this scenario, cells swell, disrupt their membranes, and release their contents, potentially leading to inflammation.

Cell Stress: Factors such as pH changes in the culture medium, nutrient deficiencies, or the presence of certain substances can increase stress within the culture environment and trigger different forms of cell death. Gelatin Coating: The consistency or homogeneity of gelatin coating may be significant; if it fails to provide sufficient support in certain areas or if there is any toxicity associated with the gelatin itself, it could lead to various degradation patterns under differing growth conditions.

Culture Conditions: Culture conditions such as room temperature, CO2 levels, or humidity can also have an impact. Inconsistencies in these factors could lead to hybrid reactions resulting in observable differences in cell death forms.

Genetic Factors: If there are genetic variations within this bEnd3 cell line or if subpopulations with distinct characteristics (e.g., variations related to stress metabolism) have arisen as a result of potential mutations or adaptations—this may explain your observations.

If your observations continue, collecting more data (such as growth rates and viability assays) would be helpful. You might consider conducting additional experiments using flow cytometry for better characterization of both types of cell death to avoid misinterpretations. Thank you for taking the time to share your findings! I hope this information helps you!

Meltem Çelik

Hello, Thank you for your kind and thoughtful advice regarding the issue I encountered. I had never considered that the cells in the second image might be swollen.. I will refer to other necrosis images as well to deepen my understanding.

I also appreciate the diverse possible causes you suggested. I will take them into account as I work to resolve this issue.

Thank you again for your help. Have a good day!

Hello,

Step 1: Maintain sterile conditions as much as possible.

Step 2: Optimize the culture conditions -37°C with 5% CO₂, concentrate on pH of the buffers, seed the cells at right density- 5 × 10³ (cells per centimeter), too high or too low leads to stress induced apoptosis

Step 3: Regular interval passage is crucial for cell growth.

Thank you for your detailed response. We typically use the bEnd.3 cell line up to passage 14. We will take your advice into consideration for our experiments.