Lung tissue was also harvested for MPO measurements. Systemic leucocyte counts Tail vein blood was mixed no with Turks solution (0.2 mg gentian violet in 1 mL glacial acetic acid, 6.25% v/v) in a 1:20 dilution. Leucocytes were identified as monomorphonuclear and polymorphonuclear cells in a Burker chamber. Serum amylase Amylase was quantified in serum with a commercially available assay (Reflotron?, Roche Diagnostics GmbH, Mannheim, Germany). MPO assay Frozen pancreatic and lung tissue were pre-weighed and homogenized in 1-mL mixture (4:1) of PBS and aprotinin 10 000 KIE?mL?1 (Trasylol?, Bayer HealthCare AG, Leverkusen, Germany) for 1 min. The homogenate was centrifuged (153 39��g, 10 min) and the supernatant was stored at ?20��C and the pellet was used for MPO assay as previously described (Laschke et al.
, 2007). In brief, the pellet was mixed with 1 mL of 0.5% hexadecyltrimethylammonium bromide. Next, the sample was frozen for 24 h and then thawed, sonicated for 90 s, put in a water bath 60��C for 2 h, after which the MPO activity of the supernatant was measured. The enzyme activity was determined spectrophotometrically as the MPO-catalysed change in absorbance in the redox reaction of H2O2 (450 nm, with a reference filter 540 nm, 25��C). Values are expressed as MPO units?g?1 tissue. Flow cytometry Blood was collected (1:10 acid citrate dextrose) from wild-type and LFA-1 gene-targeted mice. To block Fcg III/II receptors and reduce non-specific labelling samples were incubated with an anti-CD16/CD32 for 5 min.
Then samples were stained with a PE-conjugated anti-Gr-1 (clone RB6-8C5, eBioscience, San Diego, CA, USA) antibody and with a FITC-conjugated anti-LFA-1 (clone 2D7, BD Biosciences Pharmingen, San Jose, CA) antibody at 4��C for 30 min. Erythrocytes were lysed and Cells were fixed. Cells Dacomitinib were recovered following centrifugation before being analysed with a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA, USA). A viable gate was used to exclude dead and fragmented cells. After gating the neutrophil population based on forward and side scatter characteristics, LFA-1 expression was determined on cells positive for Gr-1, which is a neutrophil marker. CXCL2 levels Tissue levels of CXCL2 were determined in serum and pancreatic tissue by using double-antibody Quantikine enzyme linked immunosorbent assay kits (R & D Systems Europe, Abingdon, UK) using recombinant murine CXCL2 as standard. The minimal detectable protein concentration is less than 0.5 pg?mL?1. Histology Pancreas samples were fixed in 4% formaldehyde phosphate buffer overnight and then dehydrated and paraffin embedded. Six micrometre sections were stained (haematoxylin and eosin) and examined by light microscopy.