[Ω]) area of the membrane (0.33 cm^2 ) as described by
Katouli et al. [16].- Prior to beginning the experiment, cell monolayers are incu-
bated overnight at 37Cin5%CO 2 in serum- and antibiotics-
free GM. - Cell monolayers with stable TER are either untreated, treated
with the desired concentration(s) ofP. aeruginosa 3O-C 12 -
HSL (e.g., 100, 200, or 300μM 3O-C 12 -HSL), or exposed
to 0.6% DMSO as a diluent control (seeNotes 2and 3 ). 3O-
C 12 -HSL or DMSO is added to both the top and bottom
compartments. - At desired time points, record the TER of the cell monolayers
using an epithelial volt-ohmmeter as per the manufacturer’s
instructions (Fig.1). - To evaluate paracellular permeability in theapical to basolateral
direction, cell monolayers grown on permeable Transwell
inserts are incubated under desired experimental conditions
(as described above) in the presence of a paracellular flux tracer:
4 kDa or 10 kDa fluorescein isothiocyanate (FITC)-dextrans
(seeNote 1). Dextran tracers of varying molecular weight and
fluorophores are also available. - FITC-dextran tracers (4 kDa or 10 kDa) are dissolved in pre-
warmed (37 C) Krebs-Ringer glucose phosphate buffer
(KRG), pH 7.3 to a concentration of 10 mg/ml. - To start the experiment, transfer the Transwell insert into a
new 24-well plate containing 1 ml pre-warmed KRG buffer per
each well. - 200μl of the FITC-dextran tracer solution is added to the
apical surface of the cell monolayer (i.e., the top compartment)
and the cells are incubated at 37Cin5%CO 2 for the desired
period of time. - At desired time points, samples from the apical and basal sides
(i.e., top and bottom compartments) are transferred to a black
96-well plate and fluorescence is measured using any appropri-
ate plate reader. The excitation and emission wavelengths for
FITC-dextran fluorescence measurements should be calibrated
to 485 nm and 538 nm, respectively.
3.2 Preparation
of Whole Cell Lysates
- Caco-2 cells grown to confluence (5–8 days) on tissue culture
plates or flasks are treated with the desired concentration(s) of
P. aeruginosa3O-C 12 -HSL or 0.6% DMSO as a diluent control
in GM at 37Cin5%CO 2 for the determined amount of time
(seeNotes 2and 3 ). - Adherent cells in plates or flasks are placed on ice, washed twice
with 1 PBS pH 7.6 (in the same dish or flask), and
218 Jake Everett et al.