Horseradish peroxidase type 2

To evaluate H₂O₂ release, a horseradish peroxidase-dependent phenol red oxidation microassay was used (18 (link), 19 (link)). In this assay, two million peritoneal cells were suspended in 1 mL freshly prepared phenol red solution that consisted of ice-cold Dulbecco's PBS containing 5.5 mM dextrose, 0.56 mM phenol red (Sigma), and 8.5 U/mL horseradish peroxidase type II (Sigma). One hundred microliters of the cell suspension was added to each well and incubated in the presence or absence of 10 ng phorbol myristate acetate (PMA) (Sigma), for 1 h at 37°C in a humid atmosphere containing 5% CO₂ and 95% air. The plates were centrifuged once at 150 × g for 3 min and the supernatants were collected and transferred to another plate. The reaction was stopped with 10 μL 1N NaOH. The absorbance was measured at 620 nm with a microplate reader (MR 5000, Dynatech Laboratories Inc., Gainesville, VA, USA). Conversion of absorbance to μM H₂O₂ was done by comparison to a standard curve obtained with known concentrations of H₂O₂ (5–40 μM).

The solutions used during the CI-ELLSA assay were prepared as follows. The binding buffer consisted of 20 mM Tris-HCl buffer pH 7.4 with 0.5 M NaCl, 1 mM CaCl₂, 1 mM MgCl₂ and 1 mM MnCl₂. Concanavalin A (ConA) extracted from the jack-bean Canavalia ensiformis was used to prepare the ConA stock solution at a concentration of 0.2 mg/mL in binding buffer. The solution used to saturate the microtiter wells was prepared by dissolving BSA at 0.2 mg/mL in binding buffer. The first washing solution was phosphate-buffered saline (PBS, 2 mM Tris-HCl, 20 mM NaCl and 0.2 mM KCl, pH 7.4). The second washing solution PBS-Tween (PBS-T) was prepared as the previous one, with the addition of 0.2% Tween (v/v). The competitive solution was prepared by dissolving horseradish peroxidase (type II, 150–250 units/mg, Sigma) at a concentration of 0.1 mg/mL in binding buffer. The substrate for the peroxidase was prepared by dissolving a table set of SigmaFast OPD (o-phenylenediamine dihydrochloride) in 20 mL of ultrapure water.
The stock solutions for the standard to be used for quantification was made by dissolving 50 mg/L of invertase from baker’s yeast (S. cerevisiae invertase, grade VII, >300 units/mg, Sigma) in binding buffer.

After cell culture incubation, supernatants were removed and PACs incubated with phenol red solution [dextrose (Sigma), phenol red (Sigma), horseradish peroxidase type II (Sigma)] and plated at 37°C, 5% CO₂ for 1 h, according to Russo et al. (1989 (link)). The reaction was stopped by the addition of 1 M NaOH and the H₂O₂ concentration determined using an ELISA microreader (EL800, BIO-TEK Instruments, Inc.). The concentration was calculated using an analytical curve (0.5–8.0 nM H₂O₂).

H₂O₂ release by phagocytes was measured with the horseradish peroxidase–phenol red oxidation method described previously (Calvi et al., 2003 (link)). Briefly, phagocytes co-cultured with T. rubrum conidia or stimulated with 100 nM of phorbol-12-myristate-13-acetate (PMA; Sigma–Aldrich Co.) were centrifuged at 200 g for 10 min at 4°C and were re-suspended in 1 mL of phenol red buffer containing 50 μg/mL of horseradish peroxidase type II (Sigma–Aldrich Co.). Aliquots of 100 μL were then transferred to 96-well culture plates and were incubated for 1 h at 37°C and 5% CO₂. The reaction was stopped by the addition of 10 μL of 1N NaOH, and the absorbance was read at 620 nm (Micro-ELISA reader, Filtermax-F5). The absorbance was transformed into a standard curve of H₂O₂ that was serially diluted from 3.2 to 200.0 μM H₂O₂/mL.

Ascorbate oxidase (AAO), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), catalase, dehydroascorbic acid and dehydro-l-ascorbic acid dimer, o-dianisidine dihydrochloride and horseradish peroxidase type II were obtained from Sigma-Aldrich. AAO was dissolved as a stock at 1000 U ml⁻¹ in 50 mM succinate (Na⁺) buffer, pH 5.6, supplemented with 0.05% bovine serum albumin. Peroxidase was dissolved (1 μg μl⁻¹) and further diluted in the same buffer.
DKG was prepared from the commercial DHA by alkali treatment [56] . A stock of DHA (50 mM) was prepared in water (it took at least 30 min to dissolve DHA). A slight molar excess of NaOH (1.3 × ) was added and the mixture incubated at 20 °C for 6 min. Routinely, the hydrolysis was then stopped with 1 M l-tartaric acid and the pH was checked by pH paper (∼3.5–4.0). However, for samples to be fractionated by HPLC, hydrolysis was stopped with 1 M H₂SO₄ to a final pH of ∼1 or ∼6. Freshly-made DHA and DKG solutions were stored on ice before the assays.
DKG was prepared also by an iodate method [20] . A solution of ascorbic acid (0.12 M) was incubated with potassium iodate (0.36 M) for 5 min. KOH (1 M) was then added dropwise until the solution became colourless. Cold ethanol (8 vol, −20 °C) was added, and the precipitated DKG was vacuum filtered, rinsed in 70% ethanol, dried and stored at −80 °C.

PAC (2 × 10⁶ cells mL⁻¹) obtained as described before were maintained in RPMI-1640 culture medium at 37°C and 5% CO₂ for 24 h. At the end of the cell culture period, the supernatant was removed and macrophages were incubated with phenol red solution (dextrose (Sigma), phenol red (Sigma), and horseradish peroxidase type II (Sigma)) and plated at 37°C in 5% CO₂ for 1 h according to the methods of Russo et al. [27 (link)]. The reaction was stopped with the addition of 1 N NaOH and the H₂O₂ concentration was determined using a chemiluminescence microreader (ELx 800; BioTek Instruments Inc., Winooski, VE, USA).