Matched antibody pairs

Serum samples were obtained by cardiac puncture at the time of killing under ketamine/xylazine anesthesia. To determine whether this increased bacterial clearance was due to local cell recruitment, we analyzed the cellular content of the BAL fluid. BAL was performed with 5 mL of warm HBSS in 1 mL aliquots. One hundred microliters of the first aliquot was retained for bacterial count. The supernatant from the remainder of the first aliquot was used for cytokine analysis. The cell pellets of all aliquots were combined and counted using a Beckman-Coulter particle counter (Coulter Electronics, Danver, MA). Differential quantitation of cells was performed by counting 300 cells on cytospin slides stained with Diff-Quick (Baxter, Detroit, MI). After lavage, the lungs were sterilely transferred to 3 mL of Hanks Balanced Salt Solution and homogenized. Bacterial counts in BAL fluids and lung homogenates were determined after samples were plated in triplicate on sheep blood agar plates. Cytokines and chemokines (interferon-γ, interleukin [IL]-1ra, IL-6, IL-10, keratinocyte-derived chemokine [KC], macrophage inflammatory protein-2 [MIP-2]) were measured by sandwich enzyme-linked immunosorbent assay using matched antibody pairs (R & D Systems, Minneapolis, MN) (23 (link)).

The concentration of cytokines, the CXC chemokines KC and Macrophage Inflammatory Protein 2) and cystatin C were determined by enzyme-linked immunosorbent assay (ELISA) using matched antibody pairs (R&D Systems, Inc.) as previously described^{18 (link)}. Glypican 1 and 4 were measured using ELISA kits (Cloud Clone, Wuhan China). BUN (Blood Urea Nitrogen), AST (Aspartate Aminotransferase), and Bilirubin were measured by standard clinical methods using kits from Pointe Scientific, Inc (Canton, MI) as previously described^{15 (link)}.