Millicell electrical resistance system ers 2 volt ohm meter

Transduced and sorted (eGFP⁺) BCi-NS1.1 cells were seeded on plastic at ~3000 cells/cm² in PneumaCult-Ex Plus media (STEMCELL Technologies) and incubated at 37°C, 5% CO₂. Once cells reached 70 to 80% confluency, they were dissociated using 0.05% trypsin EDTA for 5 min at 37°C. To establish well-differentiated HAE tissue cultures grown at ALI, BCi-NS1.1 cells were seeded on 12-mm diameter transwell inserts (Corning Costar) coated with type 1 collagen (50 μg/ml) from rat tail (Corning) at ~10,000 cells/cm². Expansion media, PneumaCult-Ex Plus, was used to feed cells in both the apical and basolateral compartments until 100% confluency. After reaching confluency, the apical media was removed to transition from submerged to ALI culture, and the basolateral media was replaced with PneumaCult-ALI (STEMCELL Technologies). All cells were grown for 28 days to reach differentiation with media exchanged every other day. TEER was quantified in cultures using the Millicell Electrical Resistance System ERS-2 Volt-Ohm Meter (Millipore).

The TEER of the cultures was measured using a Millicell Electrical Resistance System ERS-2 Volt-Ohm Meter (Millipore). Fully differentiated BCI-NS1.1 cultures (n = 3) were washed immediately prior to the first TEER reading, and the collected mucus was pooled and aliquoted. The probe was sterilized in 70% ethanol for 15 minutes, then washed using PBS (no Ca²⁺ or Mg). The media was removed from the basolateral compartment of the cells at ALI, and PBS was added to both the apical and basolateral compartments and cells were left to come to room temperature. TEER measurements were conducted using the probe, including a blank cell culture insert. The final TEER measurement was determined by subtracting the TEER value of the blank insert from the TEER value of each culture insert with cells growing, then multiplying by the area of the insert (0.3 cm²). Once the first measurement had been completed, PBS was removed and media was added back to the basolateral compartment. Then, sNETs were combined in an equal volume ratio with the freshly collected mucus aliquots and 6 µl was transplanted back onto the apical surface of the cultures. Cells with the transplanted mucus were placed back into the incubator for 24 hours, then TEER was measured again. The transplanted mucus was not removed from the surface of the cultures to read the TEER after 24 hours.