Summary
A highly sensitive microspectrophotometer was developed to measure spectral changes of oxyhemoglobin (oxy Hb) in single red blood cells (RBCs) incubated with stimulated macrophages as a model of nitric oxide- (NO-) dependent cytotoxicity. Our microspectrophotometer, using a modified acousto-optic tunable filter (AOTF), allows fast spectrophotometric data acquisition. This spectrophotometer examined single human RBCs treated with various concentrations of NO that exhibited absorption spectral changes due to the conversion of oxy Hb to methemoglobin (met Hb). The change in absorption differences at α- (557–590nm) and β- (542–525 nm) bands showed a linear relationship with the concentration of NO within 100µM. We next examined RBCs incubated with murine macrophages with and without lipopolysaccharides (LPS) in the presence of glucose for 24 and 40 h. The RBCs incubated with LPS-stimulated macrophages showed significant spectral changes because of NO-dependent conversion of oxy Hb to met Hb, which suggests that RBC trap NO with very high efficiency by direct cell-cell interaction with macrophages. This spectrophotometric system is available for the use of a few drops of samples to study NO-specific cytotoxicity as a model of RBC without use of any chemical reagent in parallel with microscopic observations on cell structures such as membrane damage leading to hemolysis, adherence, and phagocytosis.
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© 1998 Springer-Verlag Tokyo
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Shiraishi, T., Tsujita, K., Kakinuma, K. (1998). Microspectrophotometry of Nitric Oxide-Dependent Changes in Hemoglobin in Single Red Blood Cells Incubated with Stimulated Macrophages. In: Ishimura, Y., Shimada, H., Suematsu, M. (eds) Oxygen Homeostasis and Its Dynamics. Keio University Symposia for Life Science and Medicine, vol 1. Springer, Tokyo. https://doi.org/10.1007/978-4-431-68476-3_69
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DOI: https://doi.org/10.1007/978-4-431-68476-3_69
Publisher Name: Springer, Tokyo
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