Cell death elisaplus

An ELISA kit that captures DNA-associated MPO was used to quantify NETs. Samples were obtained from the supernatants of treated neutrophils under different conditions or the supernatants of bronchoalveolar lavage fluid (BALF) in different groups of mice. 96-well plates were coated with anti-MPO Ab (Invitrogen, Carlsbad, CA, USA) overnight at 4°C. After washing with cold PBS, samples (20 μl) were added to the wells by mixing with 80 μl incubation buffer containing a peroxidase-labeled anti-DNA antibody (Cell Death ELISA PLUS, Roche). The plate was incubated and gently shaken for 2 hours at room temperature. Then, 100 μl peroxidase substrate (ABTS) was added and absorbance was read at 405 nm wavelength.

A capture enzyme-linked immunosorbent assay (ELISA) detecting MPO associated with DNA (MPO-DNA) was performed as described^{27 (link)}. For the capture antibody, an MPO ELISA kit (Hycult; HK210-01) and a peroxidase-labeled anti-DNA monoclonal antibody (component 2, Cell Death ELISAPLUS; Roche) were used according to the manufacturer’s directions.

Cytoplasmic histone–complexed DNA fragments were determined in islets using the Cell Death ELISAplus (Roche), per the manufacturer’s protocol. Cell pellets were analyzed to detect apoptosis. ELISA absorbance was measured at 450 nm and normalized to total DNA content (SYBRGreen; Invitrogen) using an absorbance/fluorescence microplate reader (BioTek). TUNEL was performed on transduced human islets (Apoptag In situ Apoptosis Detection Kit; Millipore) as per the manufacturer’s protocol. Human islets were prepared for staining as previously described (11 (link)). TUNEL-stained samples were counter stained with insulin and GFP antibodies. Images were captured with an IX81 microscope (Olympus) using an ORCA Flash4 CMOS digital camera (Hamamatsu). Apoptotic transduced β cells (TUNEL⁺, insulin⁺, GFP⁺) were counted as a fraction of total transduced β cells (insulin⁺, GFP⁺). DAPI served as a nuclear DNA control.

Plasma was collected by terminal heart puncture from mice anaesthetized by intraperitoneal injection of 2% avertin, using citrate (0.011 M) as an anti-coagulant. Citrated venous plasma samples from cancer patients and healthy individuals were collected at Danderyd hospital. ELISA plates (F96 Maxisorp Nunc-immuno plate; Thermo Fisher) were coated with the H3Cit antibody (ab5103; abcam) diluted 1:200 for mouse and 1:500 for human samples or the MPO antibody (HM2164-clone 266–6K1; Hycult Biotechnology) (only human samples) diluted 1:20 and incubated overnight at 4°C. Wells were washed with PBS and blocked with 3% BSA for 1 hour at room temperature. After a washing step, 20 µl undiluted mouse or human plasma were added together with 80 µl or 30 µl dsDNA-peroxidase (POD) antibody (Cell Death ELISA^PLUS; Roche) for the H3Cit and MPO ELISA, respectively, and incubated for 2 hours at room temperature. The DNA-POD antibody was diluted 1:20 for mouse, 1:100 for human H3Cit and 1:40 for human MPO detection. Following a washing step, TMB (T8665; Sigma) was added and the absorbance was measured at 650 nm with a microplate reader. Neutrophil Elastase was analyzed using a Human PMN Elastase ELISA kit (ab119553; Abcam), following instructions from the manufacturer.

To quantify ETs in cell culture supernatant, a capture ELISA to measure myeloperoxidase (MPO) associated with DNA was performed as described previously^{26 (link),47 (link)}. For the capture antibody, Mouse MPO ELISA kit (Hycult biotech, HK210–01) was used according to the manufacturer’s directions. A peroxidase-labeled anti-DNA mAb (component No.2, Cell Death ELISAPLUS, Roche; Cat. No: 11774424001) was used.