Post-mortem colon tissue was homogenised using a method adapted from processing lung tissue (Mangan et al. 2006 (link)). Levels of pro-inflammatory cytokines, such as INF-γ, IL-6, IL1-β, TNF-α, and IL-10, were detected using V-Plex Assay Plates (Meso Scale Diagnostics; Rockville, MD, USA) and assayed as per the manufacturer’s protocol. MPO activity was detected using o-phenylenediamine dihydrochloride as substrate and the data were interpolated from an MPO standard curve (Sigma). Levels of cytokines and MPO were expressed as pg per mg or U per mg, respectively, relative to colon protein (Cummins et al. 2008 (link)).
Mpo standard curve
Manufactured by Merck Group
The MPO standard curve is a laboratory equipment used to measure the concentration of myeloperoxidase (MPO) in a sample. It provides a quantitative analysis of MPO levels by generating a standard curve, which can be used to determine the concentration of MPO in unknown samples.
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4 protocols using mpo standard curve
1
Colon Tissue Cytokine Quantification
2
Colonic Cytokine and MPO Quantification
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The colonic tissue lysate was blended and homogenised as described by [23 (link)]. The expression of pro-inflammatory cytokines INF-γ, IL-6 and TNF-α, were quantified using V-Plex Assay Plates (Meso Scale Diagnostics; Rockville, MD, USA) and analyse as per the manufacturer’s. MPO activity was detected using o-phenylenediamine dihydrochloride as substrate and the data were interpolated from an MPO standard curve (Sigma). Expression of cytokines and MPO was represented as per milligram or U per milligram, respectively, relative to colonic protein.
3
Quantification of Colon MPO Activity
4
Quantification of Colon MPO Activity
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MPO activity was determined as previously described39 (link). Briefly, a colon segment (1:10 w/v) in 50 mM phosphate buffer (pH 6.0) was homogenized using an IKA T25 ULTRA-TURRAX basic homogenizer (IKA® Works, Inc., Wilmington, NC) at 4°C. The colon suspension was 10 fold-diluted with 50 mM phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide (HTAB, Sigma-Aldrich). After sonicating for 10 s and subjecting to three freeze-thaw cycles, each sample was centrifuged at 17,000 x g for 5 min. A 10 μl of each supernatant was then added to 290 μl of 50 mM phosphate buffer (pH 6.0) containing 0.167 mg/ml of o-dianisidine dihydrochlrodie (Sigma) and 0.005% H2O2, and changes in absorbance at 460 nm were measured over 5 min. The protein concentration of the supernatant sample was measured using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific). MPO activity was determined by comparison to a standard MPO curve (Sigma-Aldrich).
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