A simple approach for the analysis of intracellular movement of oxidant-producing intracellular compartments in living human neutrophils.
In human neutrophils, superoxide is generated primarily within specialized oxidant-producing intracellular compartments. The present study employs a simple methodological approach to evaluate the intracellular movement of these structures in living human neutrophils. Using a CCD camera system, we monitored fluorescence in cells loaded with the succinimidyl ester of dichlorodihydrofluorescein diacetate, which is nonfluorescent until oxidized by reactive oxygen species. Fluorescence-positive intracellular compartments became detectable after neutrophils were stimulated with phorbol myristate acetate for 1 min. Further stimulation increased the intracellular compartments in both number and size in a time-dependent manner. Upon stimulation with phorbol myristate acetate, no fluorescence was seen in intracellular compartments of neutrophils isolated from patients with X-linked chronic granulomatous disease lacking gp91-phox, a membrane component of NADPH oxidase. The method enables tracking of the movement of a single oxidant-producing intracellular compartment following cell stimulation and visualization of the intracellular structures formed by fusion of oxidant-producing intracellular compartments with endocytotic vesicles and phagosomes. Therefore, it is considered to be an informative tool for evaluation of the intracellular dynamics of oxidant-producing intracellular compartments in living human neutrophils and may have a diagnostic value.
Kobayashi
T
T
Department of Anatomy and Cell Biology, Kochi Medical School, Japan.
Zinchuk
V S
VS
Okada
T
T
Wakiguchi
H
H
Kurashige
T
T
Takatsuji
H
H
Seguchi
H
H
D001692:Biological Transport; D002451:Cell Compartmentation; D005456:Fluorescent Dyes; D006801:Humans; D008853:Microscopy; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure*; D016877:Oxidants / Q000494:pharmacology*; D013481:Superoxides / Q000378:metabolism*
D005456:Fluorescent Dyes; D016877:Oxidants; D013481:Superoxides