Hi everyone,
I am working on an experiment that requires nearly one-week incubation on 96-well plates. In this experiment, I am also measuring "time zero" to understand the cells' condition after drug treatment. But the problem is; my control cells' absorbances also decreasing in day one. I tried MTT, SRB, and DNA fluorescence. All of them were the same. Could you please explain why my control cells are dying?
(Because of my experiment procedure; the day after culturing the cells, I aspirate the media, wash the wells with PBS and add new media.)
Thank you for answers.
Hello, isn't it possible that your cell density is high? your cell condition is good? the media you are using is it optimal for your cells? I think all these different parameters are possible explanations to what you found.
To supplement Iskander's answer, I feel that your cells may not be tightly attached just yet when you rinse them with PBS. Some cell lines require longer time to attach fully to surfaces. Also, aspiration of liquid and disturbance when adding/removing/rinsing with media/PBS may cause cells to detach. Just try to be real gentle and add media/PBS through the side of the well and not directly on the cell layer. Try not to wash off the cells. Good luck!
I agree with checking the possibilities suggested by Iskander and Yi, but let me two questions: What kind of cells are you using? And, second, the day after culturing the cells before aspirate the medium, did you check under the microscope if cells showed a normal aspect or if an increased cell growth was appreciated, and most relevant, if the most cells were attached or in suspension?
Hello everyone,
Actually I have considered nearly all the things you said. I had to wash with PBS because I apply this to the experiment groups after drug treatment. I wanted the conditions to be the same.
Dear Iskander Madhi , I have repeated the experiment with different densities from 1000 to 10.000 per well. All concentrations were the same, they were all decreasing as against time zero value. My cells' conditions are also fine and I always use proper media. Thank you!
Dear Yi Jer Tan , normally my cells can easily attach even within hours. They are also epithelial. I think the day after the culturing seems fairly enough for them to adhere. Even so, I always check them under invert before wash or change the media. They seems fine. For aspiration, I use Vacusafe and be very gently. Likewise, I add everything from the side wall. But you should be right about PBS because when I check after washing, a little part of them seem apart from the layer. But I have thought that this is really a negligible factor. Thank you for your answer!
Dear María Luisa Caballero , I use epithelioid pancreatic ductal carcinoma cells. And yes, when I check them, they seem normal and attached. I think the problem could be PBS but I am not sure. I set up a new experiment to check with and without PBS again. If something come to your mind, please inform me. Thank you!
Yes, It's a good idea to rule out if PBS could be having an effect on the cells. When you perform this experiment, If you want, tell us what finally heppened!
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