A laser spectrofluorimeter for studies of cultured cells on the growth surface

Abstract

We have developed a new method for direct measurement of fluorescent probes associated with undisturbed monolayers of cultured fibroblasts. A helium-cadmium continuous wave laser produced excitation light at 325 nm to illuminate the cell monolayer on the front inner surface of a quartz sample tube. The emitted light from the cell monolayer passed through a scanning monochrometer to a low noise photomultiplier tube and was amplified with a photon-counting system. The fluorescent probe, cholesteryl 4-(3′-pyrenyl)butanoate, was incorporated into normal human low density lipoproteins (LDL). The interaction between LDL containing the fluorescent probe and the cell surface of normal human fibroblasts was examined. The uptake of the fluorescent LDL was measured as a function of temperature, concentration, time, specificity, and ability to suppress 3-hydroxy-3-methyl-glutaryl-CoA reductase. In all respects, LDL containing the fluorescent probe and native LDL were comparable. Using this technique, cell-surface interactions can be studied in situ so that changes in structure and function caused by removal of the cells from the growth surface can be avoided.