Collagenase a from clostridium histolyticum

Lung tissue was processed for flow cytometry by homogenisation with the gentleMACS tissue dissociator (Miltenyi Biotech GmbH, Germany) and digestion in medium containing 1mg/mL collagenase A from Clostridium histolyticum (Roche Diagnostics, West Sussex, UK) and 80 units/mL Deoxyribonuclease I from bovine pancreas (Sigma-Aldrich, Dorset, UK). Red cells were lysed with ACK buffer.
Lungs were lavaged 4 times via the trachea with 1.5mL of PBS containing 55mM EDTA. Lavage cells were separated by centrifugation and spun onto cytospin slides for subsequent differential staining. Blood was collected from the carotid arteries and serum separated by centrifugation using Microtainer SST tubes (BD Biosciences, Oxford, UK).
The apical lobe of the right lung was harvested from mice after lavage was performed and was stored in RNAlater buffer (Qiagen, Manchester, UK) for subsequent RNA analysis. RNA was extracted using the RNeasy mini kit (Qiagen) and cDNA generated using the Omniscript RT kit (Qiagen) and random primers.

Acetaminophen (APAP; 4-acetamidophenol), Percoll, Hepes, (heparin sodium salt grade I-A from porcine intestinal mucosa), penicillin G (sodium salt), RPMI-1640 modified media (with L-glutamine and without sodium bicarbonate and phenol red), 0.4% trypan blue solution, GSH, GSSG, S-nitrosoglutathione (GSNO), 3NT, NADH, NAD, and trifluoperazine (TFP; 10-[3-(4-methylpiperazin-1-yl)propyl]-2-(trifluoromethyl)-10H-phenothiazine), ATP Bioluminescent Assay kit, were products of Sigma Chemical Company (St Louis, MO). Collagenase A from Clostridium histolyticum was purchased from Roche Diagnostics (Indianapolis, IN). MitoSOX Red, 4-Amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM), and JC1 were obtained from Life technologies (Eugene, OR). LDH (lactate dehydrogenase) cytotoxicity detection kit was a product of from Roche Diagnostic Corporation (Indianapolis).