How to Perform a 24-Well Transwell Assay | TEER & Lucifer Yellow Validation

Unbox the 24-well transwell plates upon arrival and store them at room temperature in a dark location with parafilm wrap intact until the day of medium replacement.

Incubate plates at 37°C in 5% CO₂ for 4 hours to liquefy the shipping medium, then transfer to laminar flow hood and use a 24-well manifold to aspirate medium from apical and basal compartments. Dispense pre-warmed cell culture medium into both compartments using a multi-channel pipette.

Return apical inserts to basal compartment, replace the plate lid, and incubate in a cell culture incubator until the assay day. Change medium multiple times if assay is postponed.

Sterilize electrodes in ethanol for 5 minutes and equilibrate in pre-warmed cell culture medium for 5 minutes. Allow the plate to reach room temperature, then carefully insert electrodes into the apical and basal compartments without touching the monolayer and record resistance values in ohms for 20-30 seconds per well.

Conduct your chosen assay on the transwell plates following standard protocols. This step is at the discretion of the researcher.

Dilute Lucifer yellow stock solution in transport assay buffer, pre-warm at 37°C, and protect from light. Gently rinse apical and basal compartments with transport assay buffer (250 µL and 750 µL respectively), then remove the rinse buffer.

Add 750 µL of transport assay buffer to basal compartment and 250 µL of Lucifer yellow working dilution to apical compartment. Incubate the plate protected from light in a 37°C cell incubator for 1 hour.

Collect samples from the basal compartment and calibration curve, load them into an empty 96-well plate, and read fluorescence intensity using a fluorometer at 485/527 nm excitation/emission wavelengths.