Dear all,
I am performing Caco2 permeability (apical to basolateral) assay for several compounds. Can someone help me with the interpretation of the Papp formula: Papp = (dQ/dt)(1/(AC0))?
In particular, how do I calculate dQ/dt?
Supposing that I get the following results form the basolateral chamber (by sampling 100 ul and replacing with 100 ul of fresh medium):
Conc(t0)=0
Conc(15min)= 0.02 uM
Conc(30min)= 0.07 uM
Conc(60min)= 0.25 uM
Conc(120min)= 0.58 uM
How do I calculate dQ/dt, taking into account the volume changes for the replacement experiment?
Thank you all for your time.
Paolo
Hi Paolo. I assume that you are using lucifer yellow in your permeability assays.
Dear Dr. Stefansson,
thank you for your reply.
I am using TEER measurement and lucifer yellow to assess the monolayer integrity.
However, the data above are simply an example and are not derived from a real experiment.
I am just trying to understand how to transform the concentration of the sample into Papp value, but I find it difficult to understand what the dQ/dt is and how to calculate it.
Do you have any suggestion?
Paolo
Hi Paolo, sorry, I'm not familiar with measuring electrical resistance across a caco2 monolayer to assess permeability.
The only Caco2 permeability assay I know is spectroscopically measuring the concentrations of lucifer yellow above and below a monolayer at a fixed timepoint.
Hi Paolo:
If I understood the question, have you try to add mathematically the compound amount that have retired during sampling?
I mean, from your example (assuming a basolateral volume of 2 ml).
At 15 min, you have in the basolateral chamber 2 ml 0.02uM, which means
(2 x 10^-3 l) x 0.02 umol/l =0.00004 umol of your compound
When you take the first sample at 15 min, 100 ul 0.02 uM you take out from the chamber (100 x 10^-6 l) x 0.02 umol/l = 2 x 10^-6 umol of your compound (0.000002 umol)
At 30 min you have in the basolateral chamber 2 ml 0.07uM, which means
(2 x 10^-3 l) x 0.07 umol/l =0.00014 umol of your compound in the basolateral chamber. Taking into account the 0.000002 umol substracted in the 15 min sampling, you should have 0.000142 umol in fact.
And so on with each sampling step.
Hope this helps
Dear Dr. Lago,
thank you very much for your reply, it was absolutely helpful.
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