Single-cell [Ca2+]i analysis and biochemical characterization of pinealocytes immobilized with novel attachment peptide preparation.

Single-cell image analysis of rat pinealocytes has been difficult because they do not attach readily to coated or uncoated surfaces and typically adhere in clusters to fibroblast-like cells. In the present report, a new method for the rapid attachment of rat pinealocytes is described. Cells were prepared using papain digestion and density centrifugation and then were placed on coverslips or slides coated with PepTite-2000, a preparation containing the attachment peptide sequence Arg-Gly-Asp. Cells immobilized with this preparation responded to norepinephrine treatment with an increase in cyclic AMP and melatonin production. Single-cell analysis of Fura-2-loaded cells revealed that norepinephrine increased [Ca2+]i. This development makes it possible to conduct routine single-cell image analysis and other studies of freshly isolated rat pinealocytes.