Enzchek elastase assay kit

Following isolation, neutrophils from patients were exposed to PMA (50 nM) for 1 h, and the conditioned media with factors released by activated neutrophils (secretome) was used for NE quantification using the EnzChek^® Elastase Assay Kit (Invitrogen, Carlsbad, CA, USA, Catalog No. E12056), according to the manufacturer’s instructions.

Frozen optimal cutting temperature (OCT) sections of colonic tissues from patients and mice (8-µm thickness) were rinsed with a washing solution (2% Tween-20) and incubated at 37 °C for 6 h with BODIPY-FL-Elastin (0.5 mM) by the EnzChek Elastase Assay Kit (Invitrogen) according to a previously published protocol.^{18 (link)} As control of elastase activity, slices were pre-treated with 100 nM ELAFIN for 15 min. All sections were analyzed with Zen 2009 software (Carl Zeiss), by observers unaware of the origin and treatments. Fluorescent intensity in epithelium was quantified in at least five crypts per slide using Image J software. For each experiment, mean of fluorescent intensity from healthy controls was used as a reference to normalize data throughout the different acquisitions.

P. aeruginosa wild type (PAO1 WT) [86 (link)] and elastase-negative mutant (PAO1 ΔlasIΔrhlI) [63 (link)] were each streak-plated on a LB agar plate and incubated at 37 °C overnight. An individual colony from each plate was cultivated in LB medium at 37 °C, with shaking, overnight. Overnight cultures were diluted in 2.5 mL of ABTGC medium in six different tubes (five tubes for PAO1 WT and one tube for PAO1 ΔlasIΔrhlI) to a final optical density at 600 nm of 0.01. Each compound, at their respective concentrations, was supplemented into each of the four tubes containing PAO1 WT. All six tubes were then incubated for 24 h at 37 °C with shaking at 200 rpm. After 24 h, all cultures were centrifuged at 197.568 g for 25 min and 0.4 mL of culture supernatants were sampled from each tube. The elastase activity of the Pseudomonas aeruginosa culture supernatants was measured using the EnzChekElastase assay kit (Invitrogen, Waltham, MA, USA). The kit consists of BODIPY fluorophore (FL)-labeled DQ elastin conjugate as a substrate of elastase. The BODIPY FL-labeled DQ elastin conjugate, when cleaved by elastase enzyme, yields highly fluorescent fragments. Fluorescence was recorded every 6 min for 2.5 h using Tecan Infinite 200 Pro plate reader with excitation at 490 nm and emission at 520 nm.

Elastase activity was analyzed according to the manufacturer’s protocol using the EnzChek™ Elastase Assay Kit (E12056, Invitrogen), an assay that is well-cited in the literature [20 (link),74 (link),75 (link)]. To measure elastase activity from apical and basal cell culture supernatants, media were collected, and filter-centrifuged to concentrate before the assay. Tissue and cell lysates were prepared in 1× reaction buffer containing 0.01% Triton X. After brief sonication the lysates were centrifuged at 16,000× g for 10 min. Supernatants were collected and immediately assayed to detect elastase activity, and all assay reactions were normalized with equal protein. Pierce BCA protein assay kit (Thermo Scientific) was used for protein quantifications. Elastase activity was detected based on the fluorescence readings obtained at 485ex/535em from a Biotek Synergy H1 microplate reader.