Continuous electrochemical monitoring of nitric oxide production in murine macrophage cell line RAW 264.7

Abstract

In this study, we realized the continual and long-term electrochemical detection of NO production by stimulated macrophages using modified porphyrinic microsensor. The NO release from RAW 264.7 cells stimulated by lipopolysaccharide started 5 h after the lipopolysaccharide administration. After reaching its maximum at the sixth hour, the stable level of NO production was observed between the seventh and 12th hour of the experiment. This phase was followed by a gradual decline in NO production. A close correlation between the NO signal detected with microelectrode and nitrite accumulation, which had been determined in supernatants removed from stimulated cells, was observed. This finding was utilized for the calibration of the electrochemical experiment. The presence of iNOS enzyme, which constitutes a main requirement for NO production by stimulated macrophages, was confirmed by Western blot analysis of iNOS protein expression at key time points of the corresponding electrochemical experiment. The capability of our microsensor to instantaneously monitor the changes in the NO production by stimulated RAW 264.7 cells was demonstrated by the immediate decrease in the signal due to NO as a response to the addition of iNOS inhibitor into the cell culture medium.

Real-time electrical detection of nitric oxide in biological systems with sub-nanomolar sensitivity

“…The NO concentration reached a maximum around t ¼ 6 h and remained stable for the following 5 h, followed by a slow decrease. This time course is consistent with the expected kinetics of NO generation by iNOS in macrophage cells [46][47][48] . In contrast, when LPS (100 ng ml À 1 ) and aminoguanidine (an iNOS inhibitor; 100 mM) were added together, the change of conductance was almost negligible (Fig.…”

“…Going a step further, we have also conducted in vitro studies to test the performance of the graphene-hemin NO sensors for realtime detection of extracellular NO generated from RAW 264.7 macrophage cells. It is well known that macrophage cells can generate NO with expression of inducible NO synthase (iNOS) when stimulated by lipopolysaccharide (LPS) [46][47][48][49] . RAW 264.7 cells were seeded at 5 Â 10 5 cells per ml (cell suspension) onto the graphene-hemin device on the SiO 2 substrate (inset, Fig.…”

Asymmetric dimethylarginine regulates the lipopolysaccharide-induced nitric oxide production in macrophages by suppressing the activation of NF-kappaB and iNOS expression

“…Both of these cell lines are commonly used for experiments focused on the examination of macrophage functions under resting and activated conditions (Pekarova et al, 2009a(Pekarova et al, , 2009bViackova et al, 2010). Moreover, the difference in their origin could contribute to the amount of information about the potential universal effects of methylarginines in regulation of macrophage activation.…”

Nanostructure for Nitric Oxide Electrochemical Sensing

“…NO electrochemical sensors have been fabricated and utilized to measure NO formation and release from cells [7,8], in brain slices [9,10], in renal tubule [11], from the saphenous vein [12], and in rat hearts [13]. However, the signal transduction mechanisms of NO have not been understood yet.…”