(Millipore, Temecula, CA, USA). All primary Abs are diluted in
blocking buffer.- Secondary Abs for Fluorescence Staining: Alexa Fluor 568-
conjugated goat anti-mouse (Life Technologies, Grand Island,
NY, USA); Alexa Fluor 488-conjugated goat anti-rabbit
(Life Technologies, Grand Island, NY, USA); Atto 647 N
(fluorophore for STED) goat anti-mouse Abs (Active Motif,
Carlsbad, CA, USA). All secondary Abs are diluted in blocking
buffer. - Primary Abs for Immunoblot Analysis: anti-ZO-3 (Thermo
Fisher Scientific, Rockford, IL, USA); anti-JAM-A (Thermo
Fisher Scientific, Rockford, IL, USA). All primary Abs are
diluted in blocking buffer. Recommended dilution for primary
Abs is 1:1000 in blocking buffer. - Secondary Abs for Immunoblot Analysis: IRDye 800CW (LI-
COR Biosciences, Cambridge, UK); IRDye 680CW (LI-COR
Biosciences, Cambridge, UK). All secondary Abs are diluted in
blocking buffer: 5% nonfat milk in 1PBS pH 7.6, supple-
mented with 0.18% Tween 20. Recommended dilution for
secondary Abs is 1:10,000. - Krebs-Ringer glucose phosphate buffer (KRG): 120 mM
NaCl, 4.9 mM KCl, 1.2 mM MgSO 4 , 8.3 mM KH 2 PO 4 ,
1 mM CaCl 2 , 10 mM glucose. - For Immunoblot analysis, immunoreactive bands are visualized
by using a capable imaging system and the density ratios of the
bands are quantified by image analysis software. - Fetal bovine serum is heat treated at 55C for 30 min, ali-
quoted, and then stored at 20 C. - Dulbecco’s Modified Eagle’s Medium (DMEM) is supplemen-
ted with 10% heat-inactivated fetal bovine serum as well as
100 U/ml penicillin, 100μg/ml streptomycin, 1% nonessen-
tial amino acids, and 2 mML-glutamine. For the remainder of
this protocol, this standard culture medium will be referred to
as “growth medium” (GM). - Human epithelial colorectal adenocarcinoma Caco-2 cells
(#86010202, obtained directly from Sigma Aldrich, St.
Louis, MO, USA) are used for the experiments described
here throughout this protocol. Caco-2 cells are grown in GM
at 37Cin5%CO 2 and passaged weekly upon reaching 80%
confluence for a total of 84–95 passages. - For experiments, Caco-2 cells are grown in GM at 37Cin5%
CO 2 for 7–10 days until the cells become matured and differ-
entiated, establishing polarized epithelial monolayers on the
specified growth surface.
216 Jake Everett et al.