4 Notes
- Synthetic 3O-C 12 -HSL and 4 kDa or 10 kDa fluorescein iso-
thiocyanate (FITC)-dextrans can be purchased from Sigma
Aldrich (St. Louis, MO, USA). - If assessing the contribution of the phosphorylation state of
junction proteins on epithelial barrier integrity, cell monolayers
can be pre-treated with protein phosphatase and kinase inhibi-
tors prior to incubation with the desired quorum sensing mol-
ecule, e.g., cell monolayers can be pre-treated with 25μM
genistein (protein kinase inhibitor), 0.05μM okadaic acid
(protein phosphatase inhibitor), 0.05μM staurosporine (pro-
tein kinase inhibitor), or with all three inhibitors combined for
1 h followed with desired concentration of 3O-C 12 -HSL, or
with 0.6% DMSO as a diluent control being added to both
compartments. - If assessing the contribution of calcium signaling on epithelial
barrier integrity, cell monolayers can be pre-treated with cal-
cium blockers prior to incubation with the desired quorum
sensing molecule, e.g., cell monolayers can be pre-treated
with 0.5μM thapsigargin (sarco/endoplasmic reticulum Ca2+
inhibitor), 0.1 μM xestospongin C (inositol 1,4,5-
trisphosphate (IP(3))-induced Ca2+inhibitor), and 5μM dan-
trolene (sarcoplasmic reticulum Ca2+inhibitor) for 1 h fol-
lowed with the desired concentration of 3O-C 12 -HSL, or
with 0.6% DMSO as a diluent control being added to both
compartments. - We routinely use the Bio-Rad Detergent Compatible (DC)
protein assay (Bio-Rad Laboratories, Hercules, CA) for protein
quantification.
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