The use of fura-2 to determine the relationship between cytoplasmic free Ca2+ and oxidase activation in rat neutrophils

摘要: Abstract Incubation of rat neutrophils with fura-2-acetoxy-methyl ester ( fura-2 AM ) resulted in the loading almost exclusively into cytoplasm. Despite additional presence esterase activity granules, only 1.5% cell-associated was located within these organelles. Fura-2 leaked from at an acceptably low rate 0.16 ± 0.05% min −1 37°C. At intracellular concentrations up to 500μM, there no effect on oxidase activation; although cellular ATP content reduced approximately 50%. The peptide, f-met-leu-phe (fmlp), 1μM, produced intensity changes fluorescence excited 340nm and 380nm which were consistent a cytoplasmic Ca 2+ rise resting level 94 13nM 768 173nM (n = 6). Intracellular greater than 1mM required buffer effectively this rise, it estimated that concentration for high signal:autofluorescence ratio (100μM) buffering capacity cells increased by 10%. free induced peptide preceded activation several seconds, magnitude response dependent extent half-maximal being achieved approx. 600nM. These data therefore secondary messenger role triggering neutrophil activation.