Spectramax gemini xs spectroflurometer

BMDMs were seeded at a density 0.5×10⁵ cells per well in a 96-well plate and exposed to LPS (0.1μg/ml) after 16h. Estimation of mitochondrial membrane potential (Δψ_m) was performed using JC-10 (Enzo Life Sciences), a membrane permeable fluorescent probe. JC-10 enters selectively into mitochondria and exists as two forms, monomeric or aggregate, depending upon Δψ_m. The JC-10 monomer form predominates in mitochondria with low Δψ_m and emits in the green wavelength (525–530 nm). The JC-10 aggregate form accumulates in mitochondria with high Δψ_m and emits in the orange wavelength (590 nm). The JC-10 aggregate/monomer ratio is proportional to MMP. The final concentration of JC-10 used was 5 μM in the media and incubated for 30 min before reading the fluorescence in a SpectraMax Gemini XS spectroflurometer (Molecular Devices).

The adhesion of THP1 cells to HUVEC monolayer was measured with a fluorometric method (33 (link)). HUVEC monolayer was grown to confluency, quiesced, treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml). Wherever pharmacological inhibitors or neutralizing antibodies were used, they were added to cells 30 minutes prior to the stimulant. THP1 cells were labeled with 10 μM BCECF in serum-free medium for 30 min and the labeled cells were placed on the HUVEC monolayer at 8 × 10⁴ cells/well, and incubation continued for another 1 hr. After incubation, the nonadherent cells were washed off with PBS and the adherent cells were lysed in 0.2 ml of 0.1 M Tris-HCl buffer, pH 8.0, containing 0.1% Triton X-100. The fluorescence intensity was measured in a Spectra Max Gemini XS Spectroflurometer (Molecular Devices) with excitation at 485 nm and emission at 535 nm. Cell adhesion was expressed as relative fluorescence units.