I've been trying to assess fluorescein leakage from isolated cells plated on poly-l-lysine dishes by using fluorescein diacetate as a vehicle; however, I've noticed that shortly after I load cells, the cells appear to swell and pleb.
I've read of working concentrations from 0.1 - 10 ug/mL, and I am now at 0.1 ug/mL with these changes occurring. Cells are treated for 10 minutes at 0.1 ug/mL prior to rinsing; however at 30 - 40 minutes, there is noticeable swelling, then plebbing occurring.
I've tried searching up FDA toxicity but I have not found any papers that suggest that it should be. The FDA has been dissolved in DMSO and ideally I want to be able to monitor the cells for at least an hour with no observable changes in cell morphology.
Has anyone had this issue before? Should I further decrease the concentration?
I think most fluorophores are toxic to a point and various cell types will respond differently. DMSO is also toxic. What are the buffers you're using? What pH?
We're using HEPES buffer and the DMSO concentration is 0.01%. The cells are Lymnaea stagnalis neurons and the pH of the saline is typically ~7.9.
I am wondering if there was something potentially wrong with the dye stock as it has been dissolved and aliquoted in the fridge for ~1 year. I'm not 100% sure how stable FDA is so I wanted to figure out if this concentration should be okay for cells or if I should be looking into ordering more FDA and re-making the dye.
If the dye is in DMSO, but you're only using it at 0.01%, then that amount of DMSO is not likely to cause issues. I'm not familiar working with neurons though. It's hard to say if the FDA is still good if you didn't make it. You should be able to look at its fluorescence under microscope. Just put a drop on microscope slide to check.
The FDA definitely still works, is fluorescent, and still enters the cell; although, I was worried that it can work but still have toxicity issues. I've since decreased the working concentration from 0.1ug/mL (240nM) to 15nM such that I get no abnormal swelling.
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