Multisizer 3

Biotinylated lipid-shelled decafluorobutane microbubbles were prepared by sonication of gas-saturated aqueous lipid suspension of distearoylphosphatidylcholine (2 mg/mL), polyoxyethylene-40-stearate (1 mg/mL), and distearoylphosphatidylethanolamine-PEG (2000) biotin (0.4 mg/mL; Avanti Polar Lipids, Alabaster, AL). Surface conjugation of biotinylated ligands was performed as described previously using a streptavidin bridge.^{7 (link)} Ligands used for targeting were: dimeric recombinant murine VWF A1 domain (mature VWF amino acids 445 to 716) for targeting platelet GPIbα;^{6 (link),8 (link)} a cell-derived biotinylated peptide representing the N-terminal 300 amino acids of GPIbα for targeting endothelial VWF;^{8 (link)} and monoclonal antibodies against the extracellular domain of either P-selectin (RB40.34, BD Biosciences, San Jose, California), or VCAM-1 (clone 429, BD Biosciences). Control microbubbles were prepared with isotype control antibody (R3-34, BD Biosciences). Microbubble concentrations and size distributions were measured by eletrozone sensing (Multisizer III, Beckman Coulter).

Collected bronchoalveolar lavage (BAL) fluid was centrifuged at 1,500 × g for 3 min at 4°C and the supernatant stored at −80°C. The recovered cells were enumerated with a MultiSizer III (Beckman Coulter, Fullerton, CA) Coulter counter, a cytoslide prepared with a CytoSpin 3 cytocentrifuge (Shandon Lipshaw, Pittsburg, PA), and stained with Diff-Quik (Dade Behring, Newark, NJ) for differential counting by light microscopy. The lungs were homogenized in buffer containing 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, pH=7.4 with protease inhibitor cocktail (Calbiochem, Gibbstown, NJ) containing 500 μM AEBSF, 150 nM aprotinin, 1 μM E-64, 0.5 mM EDTA, and 1 μM leupeptin (total weight of buffer+lungs = 3 g) using a Polytron PT-2000 tissue homogenizer (Brinkman Instruments, Westbury, NY). Myeloperoxidase (MPO) was extracted from the homogenate, as previously described ^{20 (link)}.

Adipose tissue was obtained under sterile conditions and local anesthesia via periumbilical scalpel biopsy after overnight fasting as previously described (21 (link)). Two samples of 20-30 mg of tissue were immediately fixed in osmium tetroxide and incubated in a water bath at 37°C for 48 h as previously described (19 (link)), after which adipose cell size was determined via Beckman Coulter (Miami, FL, USA) Multisizer III with a 400-um aperture. In brief, 6000 cells for each duplicate sample are passed through an infrared beam which is refracted by the size of each cell. Data averaged from the duplicate samples are expressed as cell count at each cell diameter from 20-400 um, yielding a frequency histogram. Sigmaplot was used to calculate the peak center of the frequency distribution curve, which defines the diameter of mature adipocytes, without contamination by the proportion of immature smaller cells (21 (link)). This method is highly quantitative and precise since it allows for determination of the size of the mature adipocytes from the entire biopsy sample.