After inducing cells overnight by doxycycline, how can cells be detached and suspended to perform pharmacological cell based assay? Do they need to be kept serum starved for at least one hour?
When using a stable cell line for a cell-based assay, the cells should be adapted to a serum-free, chemically defined medium to avoid variability in the assay due to differences in serum composition.
To detach and suspend the cells for pharmacological assays, you can use trypsin or other appropriate dissociation reagents, following the manufacturer's instructions. Trypsin is a protease that cleaves cell surface adhesion molecules, allowing the cells to detach from the culture surface. After adding trypsin, it is important to neutralize the enzyme activity by adding an equal volume of complete growth medium. The cell suspension can then be centrifuged to pellet the cells and the supernatant removed. The cell pellet can be resuspended in assay buffer appropriate for the specific assay being performed.
Regarding serum starvation, this step may or may not be necessary depending on the specific assay being performed. Serum starvation can be used to synchronize the cell cycle and reduce the variability in the cell cycle stage among the cells in the population. However, some assays may require the presence of serum to maintain cell viability or to observe the full effects of the pharmacological agent being tested. Therefore, it is important to consider the specific requirements of the assay being performed when deciding whether or not to serum starve the cells. If serum starvation is necessary, it is typically performed for at least 24 hours, although the optimal time may depend on the specific cell line and assay.
I agree with Sergio about Trypsin. You may read more about it in my paper. Article Amphiphilic Cell-Penetrating Peptides Containing Natural and...
The most commonly used stable cell lines for cell-based assays are HeLa, CHO, and HEK293. All three cell lines are derived from cancer cells and are easily manipulated in the laboratory. They also have a long lifespan and can be used for multiple assays. Additionally, these cell lines are very robust, meaning they can withstand environmental changes without losing their ability to grow and reproduce.
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