Simultaneous monitoring of superoxides and intracellular calcium ions in neutrophils by chemiluminescence and fluorescence: Evaluation of action mechanisms of bioactive compounds in foods

Inhibition of neutrophil superoxide generation by shikonin is associated with suppression of cellular Ca²⁺ fluxes

“…The analyses were carried out using a simultaneous monitoring system for fluorescence and chemiluminescence, the CFL-C2000 (Hamamatsu Photonics K.K., Hamamatsu, Japan). The system used is the same as previously described ( 3 ) with the exception of a more sensitive photomultiplier tube (H10682-210, Hamamatsu Photonics K.K.) as a detector.…”

“…In the latter case, the anti-inflammatory compound can be an antioxidant by itself or an inhibitor of reactive oxygen species (ROS)-producing enzymes by inhibiting enzymatic activity directly or upstream events needed for enzymatic activation such as Ca 2+ fluxes. We have previously developed a simultaneous monitoring system for intracellular Ca 2+ concentration ([Ca 2+ ] i ) and superoxide anion (O 2 •− ) generation by measuring changes in fluorescence and chemiluminescence, respectively, ( 1 – 3 ) using N -formyl- l -methionyl- l -leucyl- l -phenylalanine (fMLP)-stimulated neutrophils as a tool to evaluate anti-inflammatory properties of test compounds. The fMLP-stimulated O 2 •− generation by NADPH oxidase 2 (Nox2) in cells depends on Ca 2+ -regulated pathways; ( 4 – 10 ) therefore, impairment of O 2 •− generation in parallel with altered [Ca 2+ ] i profiles will indicate that Ca 2+ fluxes are likely target candidates.…”

“…We have previously found that ibuprofen is an anti-inflammatory compound showing impaired O 2 •− generation due to altered Ca 2+ fluxes in neutrophil-like cells. ( 3 ) In contrast, if O 2 •− scavenging occurs without alterations in [Ca 2+ ] i profiles as in the case of ascorbic acid, ( 3 ) one can recognize it as an antioxidant (scavenger of O 2 •− ) not affecting upstream pathways for Nox2 activation, including Ca 2+ fluxes. Therefore, the simultaneous monitoring system is especially useful in elucidating such different anti-inflammatory mechanisms of test compounds.…”

Development and application of oxidative stress biomarkers

“…Our research group has also developed an in vitro evaluating system for the functionalities of food through optical sensing techniques using the innate immune response of HL-60 cells by the simultaneous measurement of chemiluminescence and fluorescence indicating O − 2 generation and Ca 2+ of neutrophils stimulated by agonists, respectively [48]. Using differentiated HL60 cells as neutrophil-like cells, the f-MLP was used as agonist, and it was found that AST have strong anti-inflammation activity [49].…”

Measurement of the Phagocytic Activity of Human Peripheral Blood Using a Highly Sensitive Fluorometric Detection Device Without Hemolysis

“…We have been investigating a simple prototypic device under development by Hamamatsu Photonics K.K. (Prototype) that detects microscopic amounts of fluorescence (11) and reported on a simple method of assessing the phagocytic activity of J774.1 cells (12). These cells phagocytosed pH-sensitive fluorescent particles to lower the pH level inside the phagosomes, thus making it possible to detect fluorescence at high sensitivity.…”