Proteinase inhibitor mix

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.

Five randomly chosen mice from each group were examined. Neprilysin activities were determined using a neprilysin activity assay kit (CBA079, Calbiochem, Darmstadt, Germany) according to the manufacturer’s protocols. Briefly, brains (hippocampus regions) were homogenized in 50-mM potassium phosphate buffer, pH 7.3, containing a proteinase inhibitor mix (Sigma, St Louis, MO, USA). Samples were centrifuged, and the supernatant fraction was used for neprilysin activity measurement. The hydrolysis of fluorogenic substrate peptides (2 mM Mca-RPPGFSAFKDNP-OH, R&D system, no. ES005) in 20-mM potassium phosphate buffer, pH 7.3) was measured by following an increase in fluorescence (exCitation at 320 nm and emission at 405 nm) that occurred upon peptide bond cleavage for neprilysin activity. The neprilysin activities were indicated as nanomole (obtained from a standard curve using a fluorescence unit) per microgram of protein per minute.