A simple approach to measurement of electrical parameters of cultured epithelial monolayers: Use in assessing neutrophil-epithelial interactions

Abstract

SUMMARY:Typically, electrophysiotogic studies of epithelial monolayers are either performed in formahzed Ussing chamber systems which yield highly accurate results or in simple setups using recording devices which have limitations in accuracy or the range of measurements which can be performed or both. Here we detail a simple method of interfacing traditionally accurate Ussing chambers systems with commercially available filter supports on which epithelial monolayers can be grown. We also detail simple methods for growing inverted monolayers, suitable for electrophysiologic assays, for use in studies where cells or particles must be layered by gravity on the undersurface (basolateral pole) of the filter. Both approaches allow experiments to be performed on large numbers of monolayers synchronously. As an example of the use of this system, we analyze the sequelae of neutrophil migration across monolayers of the intestinal cell line T84. Neutrophil migration across monolayers can occur in either direction, is dependent on neutrophil surface ~2 integrins, and is paralleled by a decrease in epithelial barrier function as detected electrically. We have previously shown in formal Ussing chamber studies that neutrophil-epithelial interactions elicit a modest short-circuit current indicative of electrogenic ion transport. We show here that this short-circuit current response can be readily detected using the simple approach described.

Enteropathogenic Escherichia coli dephosphorylates and dissociates occludin from intestinal epithelial tight junctions

“…Using a simplified apparatus described by Madara et al (1992), transepithelial electrical resistance was determined by passing 25 mA of current, measuring the resulting voltage deflection and applying Ohm's law (V IR) to calculate resistance.…”

Inhibition of Shigella flexneri-induced transepithelial migration of polymorphonuclear leucocytes by cadaverine

“…7. Before the addition of PMNs in this assay system, con¯uent, inverted T84 polarized monolayers (3.5´10 5 cells/well) (Parkos et al, 1991;Madara et al, 1992) were rinsed extensively in HBSS( ) to remove residual serum components. Cultures of S.¯exneri strains were prepared by washing the bacteria twice in HBSS( ) and resuspending in 300 ml buffer/10 ml culture (®nal concentration <1.5´10 9 bacteria ml À1 ).…”

“…At the end of each experiment, monolayers were cooled to 48C, and neutrophil transmigration was quanti®ed by assaying for the PMN-speci®c azurophilic granule marker myeloperoxidase, as described previously (Parkos et al, 1991;Madara et al, 1992;McCormick et al, 1993). PMN cell equivalents (CE), estimated from a standard curve, were assessed as the number of PMNs that had completely traversed the monolayer (i.e.…”

Neutrophil migration across cultured intestinal epithelial monolayers is modulated by epithelial exposure to IFN-gamma in a highly polarized fashion.

“…T84 intestinal epithelial cells (passages 70-95) were grown and maintained as confluent monolayers on collagen-coated permeable supports as previously described in detail (Dharmsathaphorn and Madara, 1990). Monolayers were grown on 0.33-cm 2 ring-supported polycarbonate filters (Costar Corp., Cambridge, MA) and utilized 6-14 d after plating as described previously (Madara et al, 1993). Inverted monolayers, used to study transmigration of PMN in the basolateral-to-apical direction were constructed as described before (Parkos et al, , 1992Madara et al, 1993).…”

“…We have previously modeled the event of PMN transmigration using human peripheral blood neutrophils and cultured, human~erived, intestinal epithelial monolayers derived from the cell line %4 (Nash et al, 1987;Parkos et al, , 1992Madara et al, 1993). Such monolayers are composed of columnar epithelial cells with features similar to those of natural crypt epithelia (Madara and Dharmsathaphorn, 1985;Madara et al, 1987).…”