This method allows comparing the transcytosis abilities of various equalized strains across polarized intestinal epithelial cells with mature junctions. This is a relatively easy technique that does not require extensive use of resources or time, and allows investigating an important step in the pathogenesis of neonatal E.coli invasive disease. Demonstrating the procedure will be Josh Wheatley, a research assistant in my laboratory.
Working inside a biosafety cabinet, seed T84 cells into polyethylene terephthalate membrane cell culture Transwell inserts. In a 24 well plate designed to hold Transwell inserts, fill the desired number of collecting wells with one milliliter of tissue culture medium or TCM with antibiotics. Seed these inserts with one times 10 to the fifth T84 cells suspended in 500 microliters of TCM with antibiotics.
Approximately 48 hours after seeding, verify with light microscopy if the monolayers have started to become confluent. Every two days after seeding, measure and record the transepithelial electrical resistance or TEER using an epithelial volt per OM meter or EVOM. Before measuring the TEER, decontaminate the electrical probe by submerging it in five milliliters of 70%ethanol for 10 to 15 minutes.
Remove the probe, shake off the excess ethanol, and let it air dry inside the biosafety cabinet for 10 minutes. Test the EVOM and probe by placing the dry decontaminated probe in a sterile well containing one milliliter of TCM with antibiotics with a sterile insert inside containing 500 microliters of TCM with antibiotics. Ensure that the EVOM reading is less than 200 OM.Record this blank value to use it in the later resistance calculations.
Remove the probe from the tube of TCM with antibiotics. Gently lower the probe into the first insert with the long electrode in the collecting well. Allow the electrode to touch the bottom of the collecting well, but do not push down as this may disrupt the epithelial monolayer and the short electrode inside the insert.
When finished, decontaminate the probe in the ethanol. Subtract the blank resistance value from each value obtained from each insert containing T84 cells. Then multiply the resulting resistance for each insert by the area of the bottom of each insert to obtain the final TEER measurement.
Once the TEER reaches 1000 OMs per square centimeter, the epithelial monolayer is mature and ready for infection assays. As the TEER matures, provide the cells with a fresh medium every one to two days. In a new 24 well plate, add one milliliter of TCM with antibiotics to one well for each seeded insert.
Using sterile forceps, carefully transfer the inserts to the newly replenished wells. Replace the media in the inserts. Remove the old media from the inserts by tilting the plate, and gently remove the media with a pipette tip along the side of the insert.
Add 500 microliters of TCM with antibiotics to the inserts and visualize the monolayer to verify that it remains intact. The day before the experiment, measure and record the TEER. Replace the medium with one milliliter of TCM without antibiotics in the plate well, and 500 microliters in the insert.
Take a labeled 15 milliliter conical tube with five milliliters of sterile lysogeny broth or LB, and use a sterile lube to inoculate the broth with one colony from one Escherichia coli strain. Incubate the culture overnight with the cap from the tube loosened. The next day, add 250 microliters from overnight LB culture to 25 milliliters of TCM without antibiotics in a 50 milliliter conical tube.
Incubate the tube for two hours. Measure the TEER across each insert. Record these as the TEERs at the time T to zero hour.
Move the inserts to wells in a new plate and change the media. Fill the new collecting wells with 500 microliters and inserts with 400 microliters of TCM without hand antibiotics. Keep the inserts inside the tissue culture incubator until the time of infection.
After two hours, remove the morning bacterial cultures from the shaker and centrifuge. Resuspend the pellet in TCM without antibiotics. Then use a spectrophotometer to adjust the optical density or OD to 0.7 or 0.9 and further dilute to a concentration of 1 million colony forming units or CFU per milliliter.
Infect each insert with 100 microliters of the OD adjusted inoculum. Note the time and record it as time zero hour. Place the inoculum bacterial suspension to quantity CFU per milliliter using the track dilution method plating 10 microliter aliquot on a square LB auger plate.
Every 30 minutes following the inoculation, fill new wells with 500 microliters of TCM without antibiotics, and transfer the inserts to these new wells. Collect the media from the used collecting well for each insert into a separate labeled tube and place the tube on ice. Return the Transwell plate to the incubator between time points.
For each insert, combine the collected media from different time points and vortex briefly. Plate the collected media on LB auger plates using the track dilution method to quantify the number of bacteria transcytose in the first two hours of the experiment. Collect the media at four and six hours.
Additionally, at six hours, plate the media from the control wells. At six hours, measure and record the TEER. After overnight incubation, count the bacterial colonies manually on the track dilution LB plates.
During the proliferation of the sterile T84 cells, the TEERs across the T84 monolayers continuously rise over time, surpassing 1000 OMs per square centimeter approximately after seven days from seeding. Transcytosis of neonatal E.coli isolates over time is shown here. The comparisons of the mean transcytosis values demonstrated significant differences between the neonatal E.coli isolates one versus two at two hours post-infection.
In contrast, the nonpathogenic E.coli strain DH5 alpha underwent minimal transcytosis. TEER results before and after infection with two neonatal E.coli clinical isolates with different abilities to transcytose intestinal epithelial cells are shown here. The rate and the amount of transcytosis vary among strains but the TEER significantly increases after infection with both pathogenic strains compared to the non-infected inserts.
Performing TEER measurements in a consistent manner is crucial for obtaining reproducible results with this method. Following strict sterile techniques to avoid contamination is also very important.
