Segments of distal colon were cut along the mesenteric border and
mounted in Ussing chambers (Physiological instruments, San Diego, CA), with a
window area of 0.09 cm2, according to protocol.60 (link),61 (link) Baseline short-circuit current (Isc) values were
obtained at equilibrium, 15 min after the tissues were mounted and expressed as
μA/cm2. Conductance (G) was also determined at baseline
as an indicator of ion flux, or paracellular permeability, and expressed as
mS/cm2. FITC labeled dextran (4 kDa, Sigma) was used as a probe
to assess macromolecular permeability, and was added to the luminal buffer at a
final concentration of 2.2 mg/mL once equilibrium was reached. Serosal samples
were taken at 30 min intervals for 2 hrs and replaced with fresh buffer to
maintain constant volumes. Fluorescence was measured by end point assay
(Victor4X, Perkin Elmer, Waltham, MA) and the flux of FITC-dextran from the
mucosa to the serosa was calculated as the average value of three consecutive
stable flux periods (30–60, 60–90 and 90–120 min) and
expressed as μg/ml/cm2/h.
mounted in Ussing chambers (Physiological instruments, San Diego, CA), with a
window area of 0.09 cm2, according to protocol.60 (link),61 (link) Baseline short-circuit current (Isc) values were
obtained at equilibrium, 15 min after the tissues were mounted and expressed as
μA/cm2. Conductance (G) was also determined at baseline
as an indicator of ion flux, or paracellular permeability, and expressed as
mS/cm2. FITC labeled dextran (4 kDa, Sigma) was used as a probe
to assess macromolecular permeability, and was added to the luminal buffer at a
final concentration of 2.2 mg/mL once equilibrium was reached. Serosal samples
were taken at 30 min intervals for 2 hrs and replaced with fresh buffer to
maintain constant volumes. Fluorescence was measured by end point assay
(Victor4X, Perkin Elmer, Waltham, MA) and the flux of FITC-dextran from the
mucosa to the serosa was calculated as the average value of three consecutive
stable flux periods (30–60, 60–90 and 90–120 min) and
expressed as μg/ml/cm2/h.