In situ measurement of degranulation as a biosensor based on RBL-2H3 mast cells.
A direct degranulation assay has been developed to enable the use of RBL mast cells as a biosensor for screening chemical libraries for drug discovery and environmental toxicity evaluation. Release of beta-hexosaminidase into the extracellular milleu is widely used to characterize cellular components and mechanisms involved in stimulated exocytosis, including those initiated by crosslinking of IgE receptors on mast cells. To adapt this versatile assay for high throughput screening, we developed a direct, in situ method in which beta-hexosaminidase detection is carried out in a single step, convenient for multi-sample processing and thus for biosensor applications. This direct assay is efficient for measuring exocytosis in antigen-stimulated RBL mast cells, detecting antigen concentrations as low as 1 pM. We also demonstrate its utility in detecting inhibition of degranulation by a known pharmacologic inhibitor that blocks Syk tyrosine kinase activity critical for cell activation.
Naal
Rose Mary Z G
RM
Baker Laboratory, Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853-1301, USA.
Tabb
Joel
J
Holowka
David
D
Baird
Barbara
B
D000818:Animals; D001681:Biological Assay / Q000295:instrumentation* / Q000379:methods; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D015550:Cell Degranulation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D004867:Equipment Design; D019544:Equipment Failure Analysis; D016231:Fluorescence Polarization Immunoassay / Q000295:instrumentation* / Q000379:methods; D008407:Mast Cells / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000032:analysis* / Q000494:pharmacology; D001619:beta-N-Acetylhexosaminidases / Q000032:analysis / Q000378:metabolism*
D012710:Serum Albumin, Bovine; D001619:beta-N-Acetylhexosaminidases