Real-time TEER of HBMEC monolayers and phase contrast microscopy.
(A) Confluent HBMEC monolayers were treated at 37°C for 24 h in the atmosphere of 5% CO2 with 1 µg/ml of either InhA (red) or Npr599 (blue). Average traces of two wells were recorded in two-minute intervals for a 24-h period. The bars show the extent of variability between independent wells. The arrow marks the addition of protease. (B) Phase contrast microscopy of HBMEC monolayers at 24 h post treatment. The arrows point to gaps formed in the monolayers of HBMECs after treatment described in (A). InhA, center panel; Npr599, right panel; mock-treated control, left panel; 100x magnification. (C) Transwell permeability assay of HBMECs treated as in (A). Positive control included treatment with B. anthracis culture supernatants (Sups) diluted 10-fold with cell culture medium in the absence (white bar) or presence (red bar) of o-phenanthroline (100 µM). Leakage of FITC-dextran across monolayers was determined as described in Materials and Methods. All measurements were made in triplicates, and the experiment was repeated twice. NT, control untreated cells.