Cytometric bead array: a multiplexed assay platform with applications in various areas of biology.

The introduction of flow cytometric bead-based technology has added a new approach for investigators to simultaneously measure multiple analytes in biological and environmental samples. This new technology allows for (1) evaluation of multiple analytes in a single sample; (2) utilization of minimal sample volumes to glean data; (3) reproducibility and results comparative with previous experiments; (4) direct comparison with existing assays; and (5) a more rapid evaluation of multiple samples in a single platform. The cytometric bead array (CBA) system enables simultaneous measurement of multiple analytes in sample volumes too small for traditional immunoassays.

The bacterial peptide N-formyl-Met-Leu-Phe inhibits killing of Staphylococcus epidermidis by human neutrophils in fibrin gels.

To study human neutrophil (polymorphonuclear leukocyte (PMN)) migration and killing of bacteria in an environment similar to that found in inflamed tissues in vivo, we have used fibrin gels. Fibrin gels (1500 microm thick) containing Staphylococcus epidermidis were formed in Boyden-type chemotaxis chambers. PMN migrated < 300 microm into these gels in 6 h and did not kill S.

Neuronal expression of a functional receptor for the C5a complement activation fragment.

The present studies were undertaken to determine whether neuronal subsets in normal brains constitutively express functionally competent C5a receptors. In situ hybridization studies coupled with immunohistochemical approaches revealed that most neurons in the hippocampal formation, many pyramidal cortical neurons, and cerebellar Purkinje neurons in normal human and murine brains constitutively express C5a receptors. Neuronal C5a receptors bound C5a-coated fluorescent microspheres, and primary rodent hippocampal neurons responded to C5a with increased calcium fluxes via a pertussis-sensitive, presumably Gi-coupled protein.

Identification of ligand effector binding sites in transmembrane regions of the human G protein-coupled C3a receptor.

The human C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor (GPCR) composed of seven transmembrane alpha-helices connected by hydrophilic loops. Previous studies of chimeric C3aR/C5aR and loop deletions in C3aR demonstrated that the large extracellular loop2 plays an important role in noneffector ligand binding; however, the effector binding site for C3a has not been identified. In this study, selected charged residues in the transmembrane regions of C3aR were replaced by Ala using site-directed mutagenesis, and mutant receptors were stably expressed in the RBL-2H3 cell line.

Cloning and preliminary pharmacological characterization of the anaphylatoxin C5a receptor in the rabbit.

1 The rabbit receptor for C5a was cloned from a genomic library and found to be 79.5% identical to the human homologue, the highest degree of similarity found so far in nonprimate laboratory animals. 2 The rabbit C5a receptor stably expressed in RBL cells binds human 125I-C5a (2 nM).