Assessing Transcytosis of E. coli Across Intestinal Epithelial Monolayers

Samenvatting

Source: Islam, A., et al., Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates. J. Vis. Exp. (2023)

This video demonstrates the procedure for assessing transcytosis of neonatal Escherichia coli across intestinal epithelial monolayers using transwell inserts, electrical resistance measurement, and bacterial quantification.

Protocol

1. Inoculation of the cells (start of the experiment)

1. After exactly 2 h, remove the morning bacterial cultures from the shaker and centrifuge them for 10 min (1,900 x g, 4 °C).
   NOTE: For all the following steps, keep all the bacterial suspensions on ice to minimize growth.
2. Resuspend the bacteria pellet in the tissue culture medium (TCM) w/o antibiotics. Use a spectrophotometer to adjust the optical density (OD) to 0.7-0.9, and further dilute with TCM w/o antibiotics to a concentration of 1 x 10⁶ colony-forming units (CFU)/mL (approximately 1:100 dilution). Use this bacterial suspension to infect each insert with 1 x 10⁵ CFU per 100 µL volume.
3. Label the transwell plate and infect each insert with 100 µL of the OD-adjusted inoculum (a total of 1 x 10⁵ CFU per insert). The assay has now begun. Note the time and record it as t = 0 h.
4. Plate the inoculum bacterial suspension to quantify the CFU/mL using the track dilution method, plating 10 µL aliquots on square LB agar plates.

2. Quantifying transcytosis

1. Every 30 min following inoculation, fill new wells with 500 µL of TCM w/o antibiotics. Transfer the inserts to these new wells using a different set of sterile forceps for each different bacterial strain.
2. Collect the media from the used collecting well for each insert into separate labeled tubes. Place these tubes on ice. In between time points, return the transwell plates to the incubator.
3. For each insert, combine the collected media from t = 0.5 h, t = 1 h, t = 1.5 h, and t = 2 h, and vortex briefly. Plate the collected media on Luria-Bertani (LB) agar plates using the track dilution method to quantify the amount of bacteria transcytosed in the first 2 h of the experiment.
   NOTE: Retrieving the bacteria every 30 minutes and keeping them on ice minimizes bacterial growth in the collecting wells, and measurements are done on predominantly transcytosis bacteria.
4. Place the labeled track dilution LB agar plates in the bacterial incubator at 37 °C without supplemental CO₂, and return the T84 transwell plate to the tissue culture incubator.
5. At t = 4 h, repeat step 2.3 by combining the collected media from t = 2.5 h, t = 3 h, t = 3.5 h, and t = 4 h.
6. At t = 6 h, repeat step 2.3 by combining the collected media from t = 4.5 h, t = 5 h, t = 5.5 h, and t = 6 h. Additionally, at t = 6 h, plate the media from the control wells.

3. End of the experiment

1. Measure and record the transepithelial electrical resistance (TEER) at the end of the experiment, t = 6 h.
2. Decontaminate the probe by submerging it in 70% ethanol for 10-15 min. If desired, discard the inserts or save/process them for additional applications. Allow the LB agar plates to incubate overnight, and disinfect and/or safely dispose of all the other used materials.
3. After overnight incubation, count the bacterial colonies manually on the track dilution LB plates to determine the inoculum amount and the amount of E. coli transcytosis. Ensure that the control plates do not show any bacterial growth.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
15 mL sterile conical tubes	MidSci	C15B
2 mL microcentrifuge tubes	Avant	AVSS2000
50 mL sterile polypropylene conical tubes	Falcon	352070
Centrifuge	Sorvall	Legend RT
Disposable inoculation loops	Fisherbrand	22363605
Dulbecco's Modified Eagle Medium (DMEM)	Gibco	11965-084
Epithelial Volt/Ohm Meter	World Precision Instruments	EVOM
Incubator shaker	New Brunswick	Innova 4080
Incubators	Thermo Scientific	51030284	Three of these are used in the method: one for sterile tissue culturing, one for infected tissue culturing, and one for bacterial incubation.
Lysogeny broth	Difco	244610
Lysogeny broth agar	IBI Scientific	IB49101
Spectrophotometer	Unico	1100RS
T84 Intestinal Cells	American Tissue Culture Collection	CCL248
Tissue culture inserts, with polyethylene trephthalate membrane, 3 µm pores, 24 well format	Falcon	353096
Tissue culture plate, 24 wells	Falcon	353504