Hi all, I am going to do a diffusion assay using FITC. My previous results were not illustrative. So, I think if I could stain it with DAPI first it would be more illustrative but I was wondering how much the staining of the cells will affect the result of diffusion assay. Does anyone have any experience?
Just to know better, what your experiment looks like:
what is the protocol of the diffusion assay?
Ad hoc I would be lesse worried about the staining of the cells if dyes for staining live cells are used (i.e. minimal/no toxic or otherwise significant effect on the cells) - this could depend of the dye concentration and I would test a range of concentrations to find the one that gives a good signal but doesn't seem to affect the cells.
Most methods for fixing cells affect their membrane integrity and kill the cells; so, the question is if this might affect your assay or not?
Thanks @Alexander Trampe, my model is similar to transwells and I am going to use FITC to assess its diffusion through a layer of cells cultured inside the transwell to the bottom of the well.
If you use common cell fixation, you will most likely affect the diffusion behaviour in your assay as you will crosslink everything inside and between cells and freeze all active processes. You could try a dye like SiR-DNA https://spirochrome.com/product/sir-dna-kit/
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