Rhodamine transport in transwells
I finished the first experiment to look at rhodamine transport across commercial transwell membranes. I was mainly interested if there would be enough transport into the basolateral chamber to measure the rhodamine with the spec.
Experimental details: both chambers in PBS and at room temperature, spinning on the rotomix, 0.5mM initial rhodamine in donor; sample volume = 100uL replaced with fresh PBS at each sample time, rhodamine measured with the spec @ 530nm.
I used Rachel’s standard curve for rhodamine in PBS to calculate uM rhodamine from my ABS values. The curve looks reasonable to me – there may have been a couple of mistakes on my part since it was the first time I’ve done the assay. Unfortunately for me, rhodamine 123 is a substrate for the P-gp efflux pumps on cell membranes, so it would be a complicated molecule to characterize in a Transwell system with cells. Rather than trying to perfect this curve, I’m going to order a fluorescent marker for paracellular transport (sodium fluorescein and/or lucifer yellow) and repeat this experiment.