Anti mpo ab

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.

To quantify NETs in mouse plasma and in cell culture supernatant, a capture ELISA based on MPO associated with DNA was applied [22 (link)]. For the capture antibody, 5 μg/ml anti-MPO Ab (Invitrogen, Carlsbad, CA, USA) was coated onto 96-well plates (dilution 1 : 500 in 50 μl) overnight at 4°C. After washing 3 times (300 μl each), 20 μl of samples was added to the wells with 80 μl incubation buffer containing a peroxidase-labeled anti-DNA antibody (Cell Death ELISA PLUS, Roche, Indianapolis, IN, USA; dilution 1 : 25). The plate was incubated for 2 hours, shaken at 300 rpm at room temperature. After 3 washes (300 μl each), 100 μl peroxidase substrate (ABTS) was added. Absorbance at 405 nm wavelength was measured after 20 minutes of incubation at room temperature in the dark. Values for soluble NET formation were expressed as fold increase in absorbance above control.

To quantify NETs in mouse BALF and in cell culture supernatant, a capture ELISA based on MPO associated with DNA was applied (46 (link)). For the capture antibody, 5 μg/ml anti-MPO Ab (Invitrogen, Carlsbad, CA, USA) was coated onto 96-well plates (dilution 1: 500 in 50 μl) overnight at 4°C. After washing 3 times (300 μl each), 20 μl of samples was added to the wells with 80 μl incubation buffer containing a peroxidase-labeled anti-DNA antibody (Cell Death ELISA PLUS, Roche, Indianapolis, IN, USA; dilution 1: 25). The plate was incubated for 2 hours, shaken at 300 rpm at room temperature. After 3 washes (300 μl each), 100 μl peroxidase substrate (ABTS) was added. Absorbance at 405 nm wavelength was measured after 20 minutes of incubation at room temperature in the dark. Results are reported as percent of WT mice BALF or healthy cell culture supernatant ± SD, arbitrarily set at 100%.