Oxiselect myeloperoxidase chlorination activity assay

Frozen lung tissue (25 mg) was powdered on dry ice and homogenized in 0.5 mL of 0.5% trichloroacetic acid. We centrifuged the lysates for 2 min at 4 °C and 8000 rpm to separate cleared supernatant from insoluble cell debris. The sample supernatants were buffered with 10× concentrated Tris-acetate buffer containing 10 µL of 0.002% xylenol blue as pH indicator. We used an ATP assay kit (Enliten, Promega, Madison, WI, USA) to estimate the ATP concentration in the supernatant by measuring in the luminescence channel of a Cytation 5 plate reader (BioTek Instruments, Inc., Winooski, VT, USA). The results are expressed in nanomolar ATP per 25 milligram of lung tissue. Tissue lysate extracted from the powdered lung tissue was also analysed using a myeloperoxidase MPO activity assay (OxiSelect™ Myeloperoxidase Chlorination Activity Assay, Cell Biolabs, San Diego, CA, USA) and according to manufacturer’s instructions. The results are expressed in microUnit per milliliter of lung tissue lysate. We used an ELISA-based carbonyl assay (OxiSelect™ Protein Carbonyl Elisa kit, Cell Biolabs, San Diego, CA, USA) to determine the protein accumulation of carbonyl modification in powdered lung tissue lysate according to the manufacturer’s instructions. The results are expressed in nM/mL of lung tissue lysate.

At the end of EVLP, the rat lung tissues were collected and flash frozen in liquid nitrogen and then stored at -80°C. Frozen lung tissues were crushed on dry ice and the powdered tissues were used to prepare the lysates. ATP content was determined from 25 mg of powdered lung tissue and extracted in 0.5 mL of 0.5% trichloroacetic acid and centrifuged at 8000 rpm for 2 min at 4°C. The supernatant was isolated, and 10X Tris-acetate was added to neutralize the pH to 7.4 with a 10 μL of 0.002% xylenol blue used as pH indicator. ATP concentration in the supernatant was determined enzymatically with an ATP assay kit (Enliten,; Promega, Madison, WI, USA) by measuring in the luminescence channel of a Cytation 5 plate reader (BioTek Instruments, Inc., Winooski, VT, USA). Tissue lysates extracted from the powdered lung tissue were also analyzed using 1) a myeloperoxidase (MPO) activity assay (OxiSelect™ myeloperoxidase chlorination activity assay, Cell Biolabs San Diego, CA, USA) and 2) an ELISA-based carbonylated proteins assay (OxiSelect™ carbonyl protein ELISA assay, Cell Biolabs San Diego, CA, USA) according to manufacturer’s instructions. A microBCA protein assay kit was used with bovine serum albumin as standard to determine protein concentration in the supernatant according to the manufacturer’s instructions (Thermo Scientific, Rockford, Il, USA).

Frozen lung tissue (25 mg) was powdered on dry ice and homogenized in 0.5 mL of 0.5% trichloroacetic acid. We centrifuged the lysates for 2 min at 4 °C and 8000 rpm to separate cleared supernatant from insoluble cell debris. The sample supernatants were buffered with 10 × concentrated Tris-acetate buffer containing 10 µL of 0.002% xylenol blue as pH indicator. Furthermore, sample pellets were reconstituted to their original volume with 1 × PBS and used to determine the protein concentrations with the Pierce microBCA protein assay kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instructions and bovine serum albumin as standard. We used an ATP assay kit (Enliten, Promega, Madison, WI, USA) to estimate the ATP concentration in the supernatant by measuring in the luminescence channel of a Cytation 5 plate reader (BioTek Instruments, Inc., Winooski, VT, USA). The results are expressed in nanomolar ATP per milligram of proteins. Tissue lysate extracted from the powdered lung tissue was also analysed using a myeloperoxidase (MPO) activity assay (OxiSelect™ myeloperoxidase chlorination activity assay, Cell Biolabs San Diego, CA, USA) and according to manufacturer’s instructions. The results are expressed in milliunits per milligram of proteins.