Ab38994

Neutrophils and monocytes were isolated from blood using density gradient centrifugation [26] (link) and positive selection for CD14 using immuno-magnetic beads (Miltenyi Biotec, San Diego, CA), respectively. Cells were lysed in radio-immunoprecipitation assay (RIPA) buffer containing protease inhibitors (at 5×10⁶ cells/ml), and frozen at −80°C. Intact neutrophils were immunostained for surface MMP-8 using Alexa 488 and rabbit anti-MMP-8 IgG (ab38994, Abcam) or non-immune rabbit IgG as a control (Dako, Carpinteria, CA) [12] (link), [17] (link) and staining quantified using a FACS Canto II flow cytometer (BD, Franklin Lakes, NJ).

MMP-8 was quantified in blood and lung samples using an ELISA (R&D Systems, Minneapolis, MN). MMP-8 levels in homogenates of lung samples were corrected for GAPDH levels which were measured in arbitrary units using a commercial kit (eBioscience, San Diego, CA). MMP-8 results were expressed as pg of MMP-8 per arbitrary unit of GAPDH. MIP-1α and IP-10 were quantified in BALF samples using ELISAs (PeproTech, Rocky Hill, NJ). MMP-8 forms were analyzed in BALF (50 microliters/sample) and lung lysates (100 micrograms of protein/sample) using western blotting [12] (link), [17] (link) and a polyclonal rabbit anti-human MMP-8 IgG [ab38994; raised against the hinge region of MMP-8 (Abcam, Cambridge, MA)] and quantified using ImageJ software [25] (link).