SPECTROCHIMICA ACTA PART A ELSEVIER Spectrochimica Acta Part A 53 (1997) 1655-1661 Carboxyfluorescein as a fluorescent probe for cytoplasmic effects of lymphocyte stimulation Meir Cohen-Kashi a, Mordechai Deutsch a,*, Reuven Tirosh a, Herzl Rachmani b, Arye Weinreb a a The Jerome Schottenstein CeNscan Center for Early Detection of Cancer, Department of Physics, Bar-Ilan University, Ramat-Can 52900, Israel b Be&son Hospital, Sackler School of Medicine at Tel-Aviv University, Petach Tikva, Israel Received 9 September 1996; accepted 27 September 1996 Abstract The application of carboxyfluorescein (CF), as an impermeable fluorescent probe for lymphocyte stimulation with phytohaemagglutinin (PHA), is investigated by following a decrease in the degree of fluorescence polarization. Since CF does not enter the mitochondria, the present results indicate that the measured effect of stimulation occurs in the cytoplasm. The results also reveal that the fluorescence yield of intracellular CF is smaller than that of extracellular CF. Moreover, the degree of fluorescence polarization of intracellular CF is inversely related to its concentration. Following cell disruption, fluorescence intensity increases and polarization decreases.These effects might indicate a weak or reversible association of intracellular CF with cytoplasmic proteins. 0 1997 Published by Elsevier Science B.V. Keywords: Lymphocytes; Stimulation; Carboxyfluorescein; Fluorescence; Polarization; Mitochondria 1. Introduction When lymphocytes into which fluorescein (F) has been introduced, are excited by polarized light, the emitted fluorescence is also partly polar- ized. Following 45 min of stimulation by phyto- haemagglutinin (PHA), there is a decrease of up to 20% in the fluorescence polarization of lymphocytes of healthy individuals and of patients with non-malignant diseases. This effect has been used for a diagnostic test of solid tumors [l-5], * Corresponding author. Tel.: + 972 353 44615; fax: + 972 353 42019. but further studies are needed to elucidate its physiological, immunological and physical basis. The aim of the work described in the present article is to find whether CF could replace fluores- cein in testing lymphocyte stimulation, for two reasons: First, CF has been reported to leak out of the cells less than fluorescein [6], its use may therefore overcome the problem of a continuous background fluorescence production, which im- pedes the kinetic determination of intracellular fluorescence polarization. Second, CF has also been reported to enter the mitochondria much less than fluorescein [7], thus it may better distinguish between stimulation effects which might occur in 1386-1425/97/$17.00 0 1997 Published by Elsevier Science B.V. All rights reserved PIIS1386-1425(97)00102-9