Discrimination of live and early apoptotic mononuclear cells by the fluorescent SYTO 16 vital dye

Accurate detection of apoptotic cells is important for the determination of cell viability. The aim of this study was to compare the sensitivity of the cell permeant SYTO 16 fluorescent dye for detecting early apoptotic mononuclear cells (MNCs) in normal donor blood with other apoptosis assays [i.e. Annexin-V, light scatter/7-amino-actinomycin-D (7-AAD) and chloromethyl-X-rosamine (CMXRos)] and to identify critical parameters for optimal SYTO 16 staining. Apoptosis was induced in normal human leukocytes from adult peripheral blood or cord blood, or the Jurkat T-lymphocytic cell line and assessed by fluorescence microscopy and flow cytometry. Dual labelling showed that SYTO 16 detected more apoptotic MNCs compared to Annexin-V. SYTO 16 staining intensity was consistent with the light scatter profiles expected of live, apoptotic and necrotic MNCs and was more objective than light scatter/7-AAD. CMXRos staining required considerable care and may not be a robust marker of apoptotic primary MNCs. For SYTO 16 flow cytometric analysis, the optimal conditions for staining 1x10(6) leukocytes were 4 nM SYTO 16 in the presence of 30 muM verapamil for 25-45 min at 37 degrees C in media containing calcium/magnesium supplemented with protein. A P-glycoprotein inhibitor, such as verapamil, and calcium/magnesium are essential for optimal loading of SYTO 16 into live MNCs and discrimination of apoptotic MNCs in normal blood samples. SYTO 16 is a sensitive, simple, inexpensive 'live cell' method for the discrimination of live, apoptotic and necrotic normal blood MNCs and is more sensitive for detecting apoptosis in these cells than Annexin-V or light scatter/7-AAD.     

Tailoring and histochemical application of fluorescent homo-dimeric styryl dyes using frozen sections: from peroxidase substrates to new cytochemical probes for mast cells, keratin, cartilage and nucleic acids

Homo-dimers of styryl dyes were chemically tailored in order to become specific cytochemical probes for use in the life sciences. Histochemical applications using fixed cryotome sections are discussed. It is concluded, that homo-dimerization of specific styryl substrates of peroxidase (PO) by way of their covalent linkage, does not necessarily lead to improved detection sensitivity of endogenous and immuno-bound peroxidase (PO) activity. In general, these dimers act less specific towards PO activity than parent monomers. Synergetic interactions of the doubled basic dye compartments with cell constituents cause a pronounced staining of further targets at the cellular level. This behavior depends on the functional groups present in each dye compartment in a crucial manner. However, by way of chemical dye tailoring centering of these initially unwanted staining properties is possible leading to novel highly fluorescent stains for mast cells, nucleic acids, keratin and cartilage tissue. Structure/staining behavior-relationships of these stains are discussed.