Lympholyte poly solution

Mouse tissue‐derived cells were isolated from cell suspension after digestion of minced tissue with 1 mg/ml collagenase IV (GIBCO), 0.5 mg/ml Dispase (GIBCO), and 1 mg/ml DNase I (Applichem). Mouse blood cells were derived from peripheral blood after red blood cell lysis. Human immune cells were derived from density gradient separated blood polymorphonuclear cell and mononuclear cell fractions using Lympholyte‐poly solution (Cedarlane), and only autologous, sample‐matched cells were used for co‐culture. Specific cell populations were enriched by either magnetic or fluorescence‐based (FACS) sorting for cell type‐specific antibodies (see Appendix Tables S1 and S3 for detailed information). Biotinylated antibodies with MACS anti‐biotin magnetic beads and MACS columns (Miltenyi Biotec) were used for magnetic sorting. FACS sorting was done on FACS‐Aria II or FACS‐ARIA Fusion and FACS analysis on LSR Fortessa machines (all BD Biosciences).

PMN were purified from EDTA-anticoagulated monkey blood by fractionation over lympholyte-poly solution (CEDARLANE, Burlington, ON) as described by the manufacturer. Naive monkey PMN (1 × 10⁶ cells) in 100 μl Hank's balance salt solution containing 0.1% globulin-free BSA and 1 mM each of CaCl₂ and MgCl₂ (Hank's balance salt solution-BSA⁺⁺) were treated with f-met-leu-phe (5 × 10⁻⁷ M final concentration) for 10 min at room temperature. Unlabelled Fab107 (75 μg ml⁻¹) was added to one-half of the replicate tubes for an additional 30 min at 37 °C, followed by addition of mouse mAb24 IgG (20 μg ml⁻¹) to all tubes and incubation for an extra 30 min at 37 °C. Cells were washed once, stained with anti-mouse Fc-specific fluorophore-labelled antibody (for 30 min at 0 °C), washed again, fixed with 1% formaldehyde in PBS and analysed using a FACS-Calibur or LSRFortessa flow cytometer (BD Biosciences, San Jose, CA). The FlowJo software was used to calculate mean fluorescence intensity. For detection of in vivo binding of mouse monoclonal antibody to monkey PMN, 1.4 μg of fluorescein isothiocyanate-anti-mouse Fc-specific monoclonal antibody (Jackson ImmunoResearch, West Grove, PA) was added to purified PMN and the mixture incubated for 20 min on ice. Cells were then washed once, fixed and analysed by flow cytometry.

Human neutrophils from healthy donors were isolated using Lympholyte®-poly solution (CL5070; Cedarlane) and stimulated with supernatants (500 μL) harvested from HT29 cells, either untreated (control; serum-free DMEM media) or treated with LPS ± LL-37 (for 4 h). Immunofluorescence analysis was performed as described above, using an anti-neutrophil elastase antibody (5 μg/mL; MAB91671; R&D Systems) and secondary Alexa549-conjugated donkey anti-mouse IgG antibody. For neutrophil elastase secretion quantification, fluorescence intensity was calculated using ImageJ 1.50i software. Average fluorescence intensity per replicate was quantified in five randomly selected fields of view and represented as fold increase in MFI.