Lung tissue was also harvested for MPO measurements Systemic leu

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.

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