Dear all,
I am performing Caco2 permeability experiments using transwell inserts and I am using phenol red permeability assay to verify the monolayer integrity.
What is the maximum phenol red permeation allowed in order to state that there is no damage to the cell monolayer?
Because phenolsulfonphthalein is a marker solute for paracellular leakage, there really is no absolute "correct" value. Apparently, few use this to profile their cell monolayer system's baseline. Many use 14C-mannitol for which I've seen apparent permeability coefficients of 0.1 - 0.5 x 10^-6 cm/sec. We used to use sulfasalazine and LC-MS and it also had Papp values in this range. Although it is more correct for each lab to statistically establish "acceptable" values, and determine what represents a high or leaky outcome while considering assay variability, you could start by setting an arbitrary cutoff of >3x your average. I would shoot for consistently measuring
Dear Dr. Thomas J Raub
thank you for your kind answer, which will be very useful to me.
Do you have by chance some reference for this? Particularly for the "consistent 5% per hour apical-to-basolateral leakage of phenol red"?
I am consistently obtaining 0.5-2% per hour apical-to-basolateral leakage of phenol red, using 500 uM in the top chamber. Should I believe my monolayer is intact? TEER values are ok.
Best regards
Take a look at the paper for an example of reported Papp for phenol red: Aungst BJ, Nguyen NH, Bulgarelli JP, Oates-Lenz K. The influence of donor and reservoir additives on Caco-2 permeability and secretory transport of HIV protease inhibitors and other lipophilic compounds. Pharm. Res. 2000, 17, 1175-1180. The apparent permeability at pH 6.5 calculates as 2x10-7 cm/s.
Alex knows this literature! You can calc Papp using your 0.2-2% per hr as follows: Papp = (Vol of donor/Area) x (0.005/3600 sec). So, using a 12-well Transwell with 0.5 mL (cm^3) in apical donor compartment and A=1.12 cm^2, you'd have a Papp of ~0.6 x 10^-6 cm/sec, which is pretty good. The upper end of 2%/h is leakier than I'd like to have.
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