I have to perform a new experiment. I have to stimulate MDA-MB-231 and THP-1 differentiated with a recombinant protein. I red that cells have to reach an 80% cell confluency before stimulation with a recombinant protein. Why is important to have an 80% confluency and not of 50% fr example? How could i see that specific cell confluency?
I think the 80% figure is probably meant to maximize the amount of cells for the sake of maximizing yield, while keeping a safety margin from full confluency, since cultured cell lines usually die when they reach confluency.
Hello Simone Marcella
The state of confluency is likely to affect the experimental results.
For instance, let us consider three cases,
1. Cells at 50% confluency means the area covered by the cells would be similar to the area not covered by the cells. Too few cells will cause the culture to grow poorly without cell-to-cell contact. This will not help to serve the purpose of your experiment.
2. Cells at 100% confluency means the entire surface of the culture vessel will be covered by cells with no space visible between them. If cells reach this level of confluence, one of two things will happen:
a. Normal cells have contact inhibition and will rarely reach 100% confluence.
b. Immortalized cells don’t have contact inhibition, which means that when they’re 100% confluent, they’ll squeeze together, and crowding will cause cells to reduce to half of their original size. At ~100% confluency the entire dish will be covered with no gaps present. Cells may be stressed and undergo cell death due to competition for resources and overcrowding of cells. So, cells at 100% confluency will again not serve the purpose.
3. Cells at 80% confluency means cells will still be growing exponentially but nearing the end of log-phase growth, which is a requirement for such an assay. They will cover most of the substratum, but gaps will still be present to avoid overcrowding. This is an ideal situation during which you may treat your cells with the recombinant protein.
I hope I have been able to answer your question!
Best.
@all Reaching a specific cell confluency before stimulation with a recombinant protein is important because it ensures that the majority of cells in the culture are in a similar physiological state, providing more consistent and reliable experimental results. While the exact confluency percentage may vary depending on the cell type and the specific experiment, 80% confluency is a commonly used guideline in many cell culture studies.
Here's why reaching an 80% confluency is often preferred:
To assess cell confluency, there are a few methods you can use:
Remember that the specific confluency requirement may vary based on the experiment and the cell type being used. It is essential to consult the literature, protocols, or experienced researchers in your field to determine the optimal confluency for your specific experimental setup. Additionally, performing some pilot experiments to optimize the cell seeding density and timing of stimulation can help you achieve the desired conditions for your study.
Ahmad Al Khraisat Malcolm Nobre Adam B Shapiro Thank you very much for your replies and your availability. Your recommendation will help me! :D
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