I am currently attempting to establish an assay to evaluate BBB permeability with hCMEC/D3 cells and I am running into an issue with attempting to disrupt the TJ proteins to cause leakage. I am seeding the cells at 30K/insert (6.5mm 0.4um) on fibronectin coated inserts and have no problem with the cells attaching and forming the monolayer after 72hrs. I am attempting to use both LPS and TNF-a at 100ng/mL each as a disruption agent for 24hrs and then assessing leakage with FITC-dextran (70kD) as a tracer molecule. The problem I am running into is I don't a big change in permeability from my control wells after testing for fluorescence from the bottom well (100uL). It's obvious the cells are there an have formed a tight monolayer as compared to the blank wells with no cells. I just cannot seem to disrupt the monolayer with any regularity and was wondering if anyone has run into this issue or might recommend a different tracer molecule.
Hi Chris Delavan . Have you tried the classical monolayer permeability assays using lucifer yellow?
Hi Chris, For the transwell assay assessing permeability, we used to make FITC dextran at a final concentration of 1mg/ml. The measurements were after every 30' and we generally measure for 6 hours after FITC administration. For fluorescent measurement, we used to pipette, 10ul of the media from the bottom of the well and dilute it with 90ul of PBS for measurement.
If you are taking 100ul of FITC dextran+media from the bottom of each well, it may have reached max fluorescence. Hope this helps.
Hello, since BBB is characterized by elevated transendothelial electrical resistance and very low cell permeability I would recommend to try TEER measurements and/or smaller size tracers.
Hi Costanza, I'm currently using a FITC-Dextran size of 70kD. What size would you recommend? I also will try out TEER measurements. We do have an endohm chamber for that I just need to get it set up and operating.
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