MPO activity was determined using the EnzChek MPO Activity Assay Kit (Invitrogen) according to the manufacturer's instructions. Briefly, the colon was homogenized in MPO assay buffer and the homogenate was centrifuged at 13000× g for 15 min at 4°C. Supernatants were used for assay. The protein concentrations in all tissue extracts were adjusted to 1 mg/ml. The absorbance at 412 nm was measured using a spectrophotometer.
Enzchek mpo activity assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The EnzChek MPO Activity Assay Kit is a fluorometric assay designed to quantify the activity of myeloperoxidase (MPO) in biological samples. The kit provides a sensitive and convenient method for measuring MPO activity without the need for specialized equipment or extensive sample preparation.
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5 protocols using enzchek mpo activity assay kit
1
Quantitative Myeloperoxidase (MPO) Assay
2
Quantifying Neutrophils in Skin Tissue
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The MPO assay was used as a surrogate marker for the presence of neutrophils in skin tissue and was carried out as described [23 (link)]. Briefly, excised pieces of skin from mice were snap frozen in liquid nitrogen and homogenized on ice in 500 μl of PBS with 0.01 M EDTA and a proteinase inhibitor mix (Sigma-Aldrich, Poole, UK) and 1 ml of 1.5% Triton X-100 in PBS. Samples were placed on a rotary shaker at 300 rpm on ice for 30 min, centrifuged at 12,000 × g for 10 min, and supernatants were collected. Total protein concentration for each sample was quantified by BCA Lowry assay (Thermo Scientific Pierce, Cramlington, UK). The protein concentration in all tissue extracts was adjusted to 0.9 mg/ml. MPO activity was determined by using the EnzChek MPO Activity Assay Kit (Invitrogen, Paisley, UK) according to the manufacturer’s instructions.
3
MPO Inhibition by Leptospira Proteins
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To test the effect of iron complementation on MPO inhibition by leptospires, 50 ng/mL purified MPO (50 µL) (R&D Systems) were incubated with increased concentrations of FeCl3 as a source of Fe3+ (0, 5, 10, 20, 40 and 100 µM) in the presence or absence of 50 µL 1 × 108 leptospires /mL for 20 min at 37°C. Leptospires were removed by centrifugation, and 50 µL of the supernatants were used for chlorination activity assay by Enzchek MPO Activity Assay Kit (Invitrogen).
To test the inhibitory effect of Leptospira membrane protein fractions after TX-114 fractionation, 50 ng/mL purified MPO (50 µL) were incubated with 10 µL of each protein fraction for 20 min at 37°C, and 50 µL of the supernatants were used for chlorination activity assay.
When recombinant proteins were employed, 50 ng/mL purified recombinant human MPO was incubated for 20 min at 37°C with the leptospiral recombinant proteins LipL21, LipL45 and Lsa63 at final concentrations of 50, 250, 500 or 1000 ng/mL. MPO incubated with purified E. coli K12 lipopolysaccharide (LPS) (Invivogen), MPO without additions or PBS alone were used as controls. Fifty microliters of the mixtures were used for chlorination activity assay.
To test the inhibitory effect of Leptospira membrane protein fractions after TX-114 fractionation, 50 ng/mL purified MPO (50 µL) were incubated with 10 µL of each protein fraction for 20 min at 37°C, and 50 µL of the supernatants were used for chlorination activity assay.
When recombinant proteins were employed, 50 ng/mL purified recombinant human MPO was incubated for 20 min at 37°C with the leptospiral recombinant proteins LipL21, LipL45 and Lsa63 at final concentrations of 50, 250, 500 or 1000 ng/mL. MPO incubated with purified E. coli K12 lipopolysaccharide (LPS) (Invivogen), MPO without additions or PBS alone were used as controls. Fifty microliters of the mixtures were used for chlorination activity assay.
4
Assessing Neutrophil Activation via MPO
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To indirectly assess neutrophil activation, the chlorination activity of released myeloperoxidase (MPO) was measured in BAL using the EnzChek MPO activity assay kit (Invitrogen), according to the manufacturer's instructions.
5
Evaluating MPO and Chlorination Activities
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MPO chlorination and peroxidase activities were determined using the Enzchek MPO Activity Assay Kit (Invitrogen), according to instructions of the manufacturer. For detection of chlorination, the kit provides nonfluorescent 3´-(p-aminophenyl) fluorescein (APF), which is selectively cleaved by hypochlorite (–OCl) to yield fluorescein. Peroxidase activity is detected using nonfluorescent Amplex® UltraRed reagent, which is oxidized by the H2O2 to form a fluorescent product. Fluorescence relative to substrate degradation were determined by excitation at 485 nm and emission at 535 nm. Supernatants from neutrophils stimulated with leptospires or bacterial culture supernatants (50 µL) were employed.
Alternatively, exudates of neutrophils stimulated with 25 nM PMA were incubated (20 min at 37°C) with L. interrogans (1 × 108, 5 × 107, 2.5 × 107 or 1 × 107 bacteria/mL), bacterial culture supernatants (at 100%, 50% or 20% dilution), PBS or EMJH. After removal of bacteria by centrifugation, chlorination MPO activity was determined.
Alternatively, exudates of neutrophils stimulated with 25 nM PMA were incubated (20 min at 37°C) with L. interrogans (1 × 108, 5 × 107, 2.5 × 107 or 1 × 107 bacteria/mL), bacterial culture supernatants (at 100%, 50% or 20% dilution), PBS or EMJH. After removal of bacteria by centrifugation, chlorination MPO activity was determined.
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