Hey everyone,
I've been struggleing with the measurement of paracellular flux in my Caco-2 monolayers for a while. I seed them into an instert for 24-well-plates (0,4µm Pores) and let them differentiate for 21d. After that I incubate them with an inflammatory cytokine mixture of TNFalpha, interferone gamma and IL-1ß in cellculture medium for 24 h. Then i wash them with HBSS, add 250 µl of FITC-Dextran (2mg/ml in HBSS) to the apical side and 750 µl HBSS to the basolateral side and incubate it for 3,5 h.
While the TER decreases by 25-40 % I'm can barely measure a FITC-dextrane flux (mw: 4 kDa). TER is also under the treshold a befriended workgroup noticed in their experiments. To exclude there is a problem with the insert itself i tested an blank insert which almost reached balanced concentrations on apical and basolateral side.
Does anyone have an idea how i can modify my assay so i might be able to measure a flux?
Do it inverse. Add stimuli in the lower compartment, measure in the upper compartment (or both). Have a positive control for >50% flux (lower molecular weight compound.
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