Ampliflu red reagent

Hydrogen peroxide (H₂O₂) production was measured using AmpliFlu Red reagent (#92001, Sigma-Aldrich). MPM cells (5x10⁴) were seeded onto a 96-well plate and allowed to grow to 90% confluence. To assess total H₂O₂ production, the medium was replaced with PBS + 2% BSA + 0.7 mM MgCl₂ and 0.7 mM CaCl₂, supplemented with 40 µM Amplex Red reagent, 1 µg/ml horseradish peroxidase, 5 µg/ml superoxide dismutase and 100 µM NaN₃. Cells were stimulated with HMW-HA (50 µg/ml), LMW-HA (200 µg/ml) and/or C1q (25 µg/ml). After 5 min, fluorescent signal was read at 576 nm with Infinite200 (TECAN).

Tyrosinase (from mushrooms, specific activity 1715 U/mg), Ampliflu™ Red reagent (1-acetyl-3,7-dihydroxyphenoxazine), DA, BSA, βLG, MeCA, l-Cys and NAC were purchased from Sigma-Aldrich (St. Louis, MO, USA). FeCl₃, phenol, 3,5-di-tert-butyl-1,2-benzoquinone (DBBQ), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), 2-mercaptoethanol (thioglycol), reduced glutathione (GSH), thioglycolic acid, n-hexane, H₂O₂, EDTA·2Na and methanol (HPLC grade) were purchased from Wako Pure Chemical Industry (Osaka, Japan). DPRA(Cys) (Ac-RFAACAA-COOH) and DPRA(Lys) (Ac-RFAAKAA-COOH) were purchased from Scrum Inc. (Tokyo, Japan). CuSO₄·5H₂O and HClO₄ were from Katayama Chemical Co. Ltd. (Osaka, Japan). NACHis, NACLeu, NACLys and dimethyl sulfoxide (DMSO) were from Tokyo Chemical Industry Co. (Tokyo, Japan). All other chemicals were of the highest purity commercially available. The highest purity Milli-Q water (Milli-Q Advantage, Merck Millipore Co., Tokyo, Japan) was used in order to avoid contamination of metal ions. The number of thiol group in BSA and βLG were determined to be 0.30 and 0.59 mol/mol protein, respectively, using the method of Elleman [51 (link)] with DTNB. The number of thiol group in BSA was close to 0.38 mol/mol BSA [30 (link)].

The H₂O₂ in the hemolymph samples as well as in the reference samples (PBS only with H₂O₂, which was added as described below) was detected using an Ampliflu^™ Red reagent (Sigma, 90101) according to [27 ]. The H₂O₂ concentration was measured in the samples collected from the insects in variants 0 and 2.
The amount of total hemolymph collected from the 5^th midstage larvae (40 μL) was dissolved in a PBS buffer pH 7.4 (460 μL). Then, each hemolymph sample was measured after mixing equal volumes H₂O₂ (to obtain a final concentration equal to 50 μM) or with PBS. One hundred μL of the reaction mixture consisted of 50 μL of the hemolymph samples and 50 μL of the compound of Ampiflu red reagent (50 μM) with horseradish peroxidase (0.1 U/mL) in a 0.05 M phosphate buffer pH 7.4. The mixture was incubated at room temperature in darkness for 30 minutes. After incubation, the absorbance was measured at 576 nm using a microplate reader (Infinite M200, Tecan). The μmol amounts of H₂O₂ were calculated according to a standard curve obtained by the incubation of the H₂O₂ solutions in the range of 0.05 to 5 μmol. Measurements were repeated at different time intervals: immediately after hemolymph collection (time 0) and 20 or 30 minutes after hemolymph incubation with H₂O₂.