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Measurement of NO production

EI Ei’ichi Iizasa
YC Yasushi Chuma
TU Takayuki Uematsu
MK Mio Kubota
HK Hiroaki Kawaguchi
MU Masayuki Umemura
KT Kenji Toyonaga
HK Hideyasu Kiyohara
IY Ikuya Yano
MC Marco Colonna
MS Masahiko Sugita
GM Goro Matsuzaki
SY Sho Yamasaki
HY Hiroki Yoshida
HH Hiromitsu Hara
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BMDMs were stimulated in RPMI-10 with 0.1 or 1.0 μg per plate of plate-coated MA or TDM in the presence of 10 ng/ml of recombinant IFN-γ for 24 h. For the assessment of TNF involvement in NO production, carrier-free recombinant mouse TNF (Biolegend #575202), LEAF purified anti-mouse TNF-α antibody (MP6-XT22; BioLegend #506331), or LEAF purified Rat IgG1 κ Isotype Ctrl antibody (BioLegend #400457) were added to the culture. The culture supernatants were collected and mixed with Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, and 2.5% phosphoric acid) at a 1:1 ratio, reacted for 10 min, and the absorbance at 550 nm was measured by a VERSAMax microplate reader using SoftMaxPro software. The nitrite concentration was calculated according to the standard curve.

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