Working on Caco 2 cells. I want to know about the cells grown on collagen for differentiation. Also, can I perform a lucifer yellow assay for assessing the permeability followed by western blot in the same cells.
Thank you
For confluence I would use a GAPDH smart flare and you can then visualise the cells using fluorescent light depending on the flare. Also smartflare is non toxic and can be added to media when media changing. In regards separation from the collagen maybe the beat way would be a collagenase digest unfortunately this is destructive to the scaffold.
I totally agree with Dr. William Richard Webb opinion.
To Dr. Bhaskar Vemu
What kind of molecular markers would you use as the differentiation of Caco-2 cells on collagen?
I recommend you should check whether CD44 (especially CD44 variant 8-10) expression is enhanced or not.
You could use an ester based cell permeant dye like Calcein AM and look under fluorescence marker. If this is not permissive, then you could probably try dextran blue of something like that to see if it passes through or not. Confluent Caco-2 monolayer should have well formed tight junctions and prevent dextran from passing through.
As for cell lysis for Western blotting, is it only western blotting you want, or there will be intermediate biochemical steps. Because if it is straight WB, I would say just use Laemmli buffer.
Thank you everyone. I am planning only western blot. Also, as a marker of differentiation I would use alkaline phosphatase activity as well as histology to visualise the cellular microvilli formation. I like the idea of smartflare. But, how accurate is a visual appearance correlation to permeability. I was thinking to rather use Fluorescent dextran at 4KDa to test the permeability. I hope it is not toxic to cells
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