Multisizer 4 analyzer

The total cell numbers, alveolar macrophages and polymorphonuclear leukocytes (PMNs) recovered by BAL were determined using a Beckman Coulter Multisizer 4 Analyzer (Beckman Coulter Indianapolis, IN, USA). Cells were differentiated using a Cytospin 3 centrifuge (Shandon Life Sciences International, Cheshire, England). Cell suspensions (1 × 10⁵ cells) were spun for 5 min at 72 × g and pelleted onto a slide. The cytospin preparations were stained with modified Wright–Giemsa stain, and cell differentials were determined by light microscopy. Differential cell counts were calculated by multiplying the total cell counts (~200 per rat) by the cell differential percentage obtained from the cytospin preparations. Using the acellular fraction of BALF, lactate dehydrogenase (LDH) activity, an indicator of general cell damage and toxicity, was measured. LDH activity was determined by measuring the oxidation of lactate to pyruvate coupled with the formation of NADH at 340 nm. Measurements were performed with a COBAS C111 auto-analyzer (Roche Diagnostics USA, Indianapolis, IN, USA).

Total cell numbers were determined using a Beckman Coulter Multisizer 4 Analyzer (Beckman Coulter, Indianapolis, IN). Polymorphonuclear leukocytes (PMN) and alveolar macrophages (AM) counts were also determined. Cells were differentiated using a Cytospin 4 centrifuge (Shandon Life Sciences International, Cheshire, England). Briefly, cell suspensions (2 × 10⁵) were spun for 5 min at 72 × g and pelleted onto a slide. Two hundred cells/rat were then counted following staining with modified Wright-Giemsa stain and the relative abundance of lung AMs, PMNs, lymphocytes, and eosinophils were determined.
Acellular LDH activity from BAL was measured to determine general cell damage and toxicity using a COBAS C111 analyzer (Roche Diagnostic Systems, Montclair, NJ).