Real‐Time Amperometric Analysis of Reactive Oxygen and Nitrogen Species Released by Single Immunostimulated Macrophages

Abstract

Macrophages are key cells of the immune system. Immunologically activated macrophages are known to release a cocktail of reactive oxygen and nitrogen species. In this work, RAW 264.7 macrophages were activated by interferon-gamma and lipopolysaccharide, and the reactive mixture released by single cells was analyzed, in real time, by amperometry at platinized carbon microelectrodes. In comparison with untreated macrophages, significant increases in amperometric responses were observed for activated macrophages. Nitric oxide (NO*), nitrite (NO2*-), and peroxynitrite (ONOO-) were the main reactive species detected. The amounts of these reactive species were quantified, and their average fluxes released by a single, activated macrophage were evaluated. The detection of ONOO- is of particular interest, as its role and implications in various physiological conditions have been widely debated. Herein, direct evidence for the formation of ONOO- in stimulated macrophages is presented. Finally, the presence of 1400W, a selective inducible nitric oxide synthase (iNOS) inhibitor, led to an almost complete attenuation of the amperometric response of activated RAW 264.7 cells. The majority of the reactive species released by a macrophage are thus likely to be derived from NO* and superoxide (O2*-) co-produced by iNOS.

Nanoelectrodes for determination of reactive oxygen and nitrogen species inside murine macrophages

“…Similarly, due to its rapid reaction with O 2 , NO was generated in situ by controlled decomposition of DEANONOate (see experi- mental section) (23). The instability of these species was not, however, an issue for in vivo experiments where ROS/RNS are produced and detected on a much shorter experimental time scale (seconds) (6)(7)(8).…”

“…The difference in sensitivity was not critical for our purpose because these electrodes were not used to measure ROS/RNS concentrations, but to collect their fluxes produced either inside (nanoelectrodes) or outside (microelectrodes) stimulated macrophages. The important parameters here are the time durations (t 1∕2 ) and the integral of the current-time responses [i.e., charge, Q, which reflects the quantity released (6)(7)(8)] recorded at the platinized electrode (Table 1). …”

“…•− and NO (5)(6)(7)(8). However, the concept that ROS and RNS released inside phagolysosomes may diffuse across the vacuole membrane and leak in the cell cytoplasm remains controversial (9)(10)(11)(12).…”

Electrochemical sensor for the detection of multiple reactive oxygen and nitrogen species from ageing central nervous system homogenates

“…Electroanalytical approaches offer similar sensitivity, spatial and temporal resolution as these imaging based approaches, but, importantly, are able to simultaneously detect multiple identified ROS/RNS species in a single measurement (Amatore et al, 2008;Amatore et al, 2010).…”

“…These are hydrogen peroxide (H2O2), peroxynitrite (ONOO -), nitric oxide (NO • ) and nitrite (NO2 -). The developed sensor was fabricated using multiwall-carbon nanotube (MWCNT) composite electrodes and modified by coating it with platinum black (Supplementary figure 1) using a previously published technology to enable the voltage-dependent detection of specific ROS/RNS (Amatore et al, 2008;Amatore et al, 2010). The sensors have been characterised for the detection of the four ROS/RNS in biological samples (see supplementary information for details on fabrication and characterisation of the sensors).…”