Act diff haematology analyser

A complete blood count (CBC) was determined using an Ac·T diff haematology analyser (Beckman Coulter, California, CA, USA), according to the manufacturer's instructions. Beckman Coulter 4C© Plus Tri‐Pack Cell Controls (Beckman Coulter) were used to confirm instrument accuracy and precision.

Bronchoalveolar lavage (BAL) samples were collected from the lower lobe of the left lung at baseline, pre-LPS infusion (or sham) (T1), post-endotoxemia/pre-resuscitation (T2) and 0 and 12 h post-saline resuscitation for ELISA analysis and aliquots cytospun onto slides for inflammatory cell counts. Arterial blood samples were collected for flow cytometry at baseline, T2, 0, 2 and 12 h post-saline resuscitation. Full blood counts were performed using the veterinary mode of the AcT diff™ haematology analyser (Beckman Coulter Australia Pty Ltd., NSW, Australia). Blood films were prepared, stained with Quick Dip (POCD Scientific, NSW, Australia) and used for a manual white blood cell differential count. Neutrophil numbers were calculated based upon the total white cell count from the AcT diff™ and the white blood cell differential count. Tissue samples were removed for histopathology, gene and protein expression studies. Pulmonary oedema was determined by measuring the wet-to-dry-weight of post-mortem left and right upper and lower lobe lung tissue. Serum albumin and total protein were assessed using commercially available kits on the COBAS Integra 400 blood chemistry analyser (Roche Diagnostics, Australia) while total protein was performed on BAL samples using the BCA (bicinchoninic acid) protein assay kit (Pierce Technology, USA).