Spectramax i3x plate reader

HEK-hTLR2 cells were plated at 7.5 × 10⁴ cells/well in 96-well plates containing DMEM + 10% heat-inactivated FBS without antibiotics (n = 3 samples per condition). The next day, the cells were treated with EVs in DMEM cell culture media for 24 h. For detection of a nuclear factor kappa-B (NF-κB) response (SEAP reporter), cell culture supernatants were incubated with QUANTI-Blue solution (Invivogen) for 1 h, pictures were taken of the plate, and absorbance was read at 630 nm on a SpectraMax i3x plate reader (Molecular Devices). For detection of an IL-8 response (Lucia luciferase reporter), cell culture supernatants were mixed with QUANTI-Luc solution (InvivoGen), and luminescence was read immediately on a SpectraMax i3x plate reader. Additionally, cell death was measured as described above.

HEK-hTLR2 cells were plated at 7.5 × 10⁴ cells/well in 96-well plates containing DMEM + 10% heat-inactivated FBS without antibiotics. The next day, the cells were treated with either live L. crispatus or G. vaginalis (10⁴–10⁶ CFU/well) or 10% (v/v) bacteria-free supernatants (10⁷–10⁵ CFU/mL culture density) in DMEM cell culture media for 24 h. In additional experiments, the cells were pre-treated with the TLR2 neutralizing antibody, anti-hTLR2-IgA (InvivoGen), for 1 h prior to exposure to live bacteria or supernatants. In these experiments, the TLR2 agonist FSL-1 (10 ng/mL, Sigma-Aldrich, St. Louis, MO) was used as a positive control for antibody efficacy. For detection of a nuclear factor kappa-B (NF-κB) response (SEAP reporter), cell culture supernatants were incubated with QUANTI-Blue solution (Invivogen) for 1 h, pictures were taken of the plate, and absorbance was read at 630 nm on a SpectraMax i3x plate reader (Molecular Devices). For detection of an IL-8 response (Lucia luciferase reporter), cell culture supernatants from the same experiment were combined with QUANTI-Luc solution (Invivogen), and luminescence was read immediately on a SpectraMax i3x plate reader. Additionally, cell culture supernatants were used in cell death assays as described below.