ALine Modular Device: Sampling Method for Small Molecule Permeability

  1. Add 100 µl of desired fluorescent small molecule at the desired concentration in medium to the top well of mod-µSiM device. Include a cell-free, coated control.
  2. Incubate at 37°C, 5% CO2 for 1 hr.
  3. Following incubation, sample media from the bottom channel and transfer to a black, flat bottomed 96-well plate (see images and video below).
    1. To sample from the channel, first remove media from the top well to stop the diffusive process. Then, draw 50 µl media into a pipet tip and lodge into one port of µSiM to serve as reservoir. Fit an empty pipet into the second port and draw a total 50 µl out of the channel. To sufficiently clear all dye from channel, sample 25 µl twice, with a couple minutes in between each wash (not shown in video). Pool washes in a black, flat bottomed 96-well plate for a total of 50 µl/well. Sampling Method Movie
  4. Prepare a reference ladder with the fluorescent small molecule. We traditionally prepare as follows: 200-, 100-, 50-, 25-, 12.5-, 6.25-, 3.125- and 0-μg/ml in medium. Transfer reference samples in 50-μl aliquots in triplicate to the 96-well plate.
  5. Measure fluorescence intensity at the appropraite Ex/Em dye used in a microplate reader.
  6. Calculate total permeability using the following equation:
    where Ct is the concentration of fluorescent small molecule in the abluminal chamber (bottom channel) at time t (determined by reference ladder), V is the volume in plate (0.050 ml), Ci is the initial concentration of fluorescent small molecule added to the luminal chamber (top wwell), t is time (in either seconds or min depending on desired units), and A is the membrane area (usually 0.014 cmbut this should be measured using imageJ).
  7. Calculate endothelial permeability (Pe) using the following equation:
    where Pt is total permeability as calculated above and Pc is permeability across the coated-control device.