Fmax fluorescence plate reader

The 3 micron pore size Neuro Probe ChemoTx® system consisting of a 96-well microtiter plate was used to assess neutrophil migration (Frevert et al., 1998 (link)). Each upper well was loaded with 4 × 10⁴ calcein-labeled neutrophils in 20 μl of the appropriate media. Treatment groups were tested in triplicate. HBSS⁺⁺ was used for chemotaxis; while HBSS⁺⁺ with chemoattractant (concentration equal to the bottom wells) was used to measure chemokinesis. Cells were allowed to migrate for 1 h at 37 °C toward lower wells containing HBSS⁺⁺, HBSS⁺⁺ with chemoattractant (10 nM LTB₄ or 10 nM PAF), or HBSS++ with VC (EtOH). Following the hour migration, non-migrated cells were removed from the top of the membrane with a cell scraper. Following centrifugation at 1000 rpm for 1 min, fluorescence of the bottom wells was measured (485 nm excitation, 530 nm emission) using an fMax fluorescence plate reader (Molecular Devices). Percent migration was calculated by dividing the fluorescence of the experimental bottom wells by the fluorescence of bottom wells containing 4 × 10⁴ cells.

To assess fMLF mediated adhesion, 1.2 × 10⁵ calcein-labeled neutrophils (60 ul) from the various treatment groups were aliquoted into individual wells of Immulon2HB flat bottom 96-well plates (Thermo Fischer Scientific) previously coated with 5% FCS in sterile PBS, as previously described [12 (link)]. The plates were then placed in a 37°C incubator while the cells settled for 10 minutes. Following the addition of fMLF (100 nM final concentration) or vehicle control (DMSO) the plate was floated in the 37°C water bath for 3 minutes. Following incubation, neutrophil adhesion was assessed using an fMax fluorescence plate reader (Molecular Devices). After an initial fluorescence reading (485 nm excitation, 530 nm emission) the plates were gently dumped in a single inverted motion; the wells were filled with 150 ul sterile PBS and another fluorescence reading was obtained. This procedure was repeated for a total of three washes to remove non-adhered cells. Fluorescence after each washing was divided by the initial fluorescence to calculate percent adhesion. The first wash that demonstrated 10% adhesion of non-stimulated cells or less (second wash on average) was considered the final result. Treatment groups were tested in triplicate.