Lsrii fortessa cell analyser

The chemotactic effect of supernatants collected from stimulated lung tissue models was assessed using a Transwell migration assay. The supernatants collected from stimulated lung tissue models (diluted 1:50 in RPMI 1640 complete media) were added to the outer chamber of Transwell plates (Costar, Corning Inc.). CXCL8 (25 and 100 ng/ml) was used as positive control. Neutrophils were seeded at 5×10⁵ cells/well in the upper chamber of a 24-well Transwell plate and incubated for 2 h at 37°C. To quantify cellular migration, cells were collected and mixed with a fixed amount of Count Bright absolute counting beads (Molecular Probes). Analysis was done using a BD LSRII Fortessa cell analyser (BD Bioscience) with gating on neutrophils and beads, respectively, to obtain the total number of cells that had migrated. FlowJo software version 9.5.3 (Tree Star) was used for flow cytometry analyses.

Chemotactic effect of HMGB1 was measured using a transwell-based assay as detailed in Berthelot et al. (2012 (link)). Whole-blood was chosen as a previous report showed that neutrophil isolation methods might affect cell motility (Sroka et al., 2009 (link)). In short, 600 μl of PBS with 1% human serum and recombinant HMGB1 or IL8 (R&D Systems, Minneapolis, MN; both supplied in preparations with endotoxin levels <1 EU/μg) was added to the basolateral side of a 3 μm pore-size polycarbonate transwell membrane (Coring incorporated, Corning, NY). One hundred microliters of human whole blood diluted 1/10 in PBS was added to the membranes apical side, and cells left to migrate for 2 h in 37°C. Migrated cells were collected and stained with anti-human CD14 PE and anti-human CD15 FITC (BD Bioscience, San Jose, CA). The number of migrated neutrophils was determined using a BD LSRII Fortessa cell analyser (BD Bioscience, San Jose, CA) in combination with CountBright Absolute Counting Beads (Molecular Probes Inc., Eugene, OR), and analyzed using the FlowJo software version 9.5.3 (Tree Star, Ashland, OR).