Celllytic m lysis buffer

NE is a serine protease found primarily within the azurophilic granules of the neutrophil [35 (link)]. To confirm neutrophil influx into the lungs, we examined total NE abundance by DuoSet ELISA according to the manufacturer’s protocol (catalogue # DY4517-05; R&D Systems). Briefly, frozen lung tissue was homogenized in CellLytic M lysis buffer with proteinase inhibitor (Sigma). Wells of a 96-well plate were incubated with capture antibody overnight at room temperature and then washed three times with PBS and 0.05% Tween (PBST). Wells were blocked with PBS and 1% bovine serum albumin (BSA) for 1 h, washed with PBST, and incubated with lung sample or standard (12.5 − 800 pg/mL) overnight at 4 °C. Wells were again washed with PBST and then incubated with detection antibody. After 2 h, wells were washed with PBST and then incubated with streptavidin conjugated to horseradish peroxidase (HRP) for 20 min. Wells were once again washed with PBST, and TMB substrate was then added to each well and incubated for 20 min. The reaction was stopped using 1 M H₂SO₄ and plates read at 450 nm using the Model 680 microplate reader and software version 5.2.1 (Bio-Rad).

Production of reactive oxygen species (ROS) was monitored spectrofluorometrically on mouse fibroblasts Balb 3 T3 using the 2’,7’-dichlorofluorescein diacetate (DCFH-DA) assay, as described by Tobi et al. [16 (link)].
In details, cells were cultivated and treated for 16 h with 1, 5, and 10 mg/ml of Mct and Mf as described above. H₂O₂-stressed cells were used as control; DMEM (2.5% CBS) was used as culture medium. After the incubation with Mct and Mf, the medium was removed and washed twice with phosphate buffer saline (PBS). Cells were treated with DCFH-DA dissolved in DMSO (final concentration 100 μM) for 30 min at 37°C, in the dark.
Cells were then treated for 2 h at 37°C in the dark with 100 μl of pre-warmed DMEM (2,5% CBS) containing 400 μM H₂O₂. At the end of the treatment, cells were washed twice, lysed with Cell Lytic M lysis buffer (Sigma Aldrich), added with 1% protease inhibitor cocktail (Sigma Aldritch) and transferred into a black 96-well plate. Fluorescent 2’,7’-dichlorofluorescein (DCF) was read fluorometrically using a Fluoroskan Ascent FL Microplate Fluorescence Reader (Thermo Scientific) at excitation and emission wavelengths of 485 and 538 nm, respectively. Each experiment was carried out in triplicate.

Protein isolation from cell culture experiments, SDS-PAGE, and Western Blot were described elsewhere^{35 (link)}. The method involves cell lysis with CellLytic M lysis buffer (Sigma-Aldrich), protein separation using ClearPAGE 10% sodium dodecylsulfate polyacrylamide gels (C.B.S. Scientific), and sample transfer to PVDF membranes (Fisher Scientific). For detection of PLA2R1, primary recombinant rabbit monoclonal antibody (AB; ab211573, Abcam, Cambridge, UK) and a secondary horseradish peroxidase conjugated goat anti-rabbit IgG (sc-2004, Santa Cruz) were used. Actin was detected using primary mouse monoclonal AB (MAB1501R, Merck Millipore, Darmstadt, Germany) and secondary horseradish peroxidase conjugated goat anti-mouse IgG (sc-2005, Santa Cruz). Protein isolation, SDS-PAGE and Western Blot were repeated three times using separate cell culture passages (biological n = 4).