Mpo elisa

For the ELISA, the supernatant of the co-cultures was collected after 24 h and stored at -20°C until further use. Supernatant from neutrophil cultures were analyzed for MPO and MMP-9. Therefore, MPO ELISA from R&D Systems (Catalog Number: DY3667) and MMP-9 ELISA from R&D Systems (Catalog Number: DY6718) were used according to manufacturer’s instructions. Briefly, capture antibody for MPO or MMP-9 was diluted in PBS (1/180) and added to 96-half area plates and incubated over night at 4°C. Afterwards, plates were washed three times using PBS with 5% Tween and plates were blocked with PBS/1% BSA for 2 h at RT. Blocking was removed and a serial dilution of MPO or MMP-9 standard, PBS/1% BSA as a negative control and diluted (1/2 for MMP-9 and 1/20 for MPO) supernatant was added to the plates for 2 h at RT. Plates were washed 5 times with PBS and 5% Tween for 1 min each and the detection antibody (1/180 diluted in PBS/1% BSA) was added and incubated for 2 h at RT. Plates were washed 5 times and Strep-HRP diluted in PBS/1% BSA was added and incubated for 1 h at RT. Detection for all ELISAs was done by the addition of TMB until color change was observed, the reaction was stopped using 1 M H₂SO₄ and plates were measured using the SpectraMax 190 plate reader (Molecular Devices LLC) at 450 nm.

An 18G catheter (BD, MA) was used to gain entrance into the trachea, and the lungs were flushed four times with a total volume of 2mL of HBSS supplemented with 0.3% FBS and 300μM EDTA. Collected bronchial alveolar lavage (BAL) was transferred into a 15 mL conical falcon tube, and placed on ice for further processing. BAL samples were separated into three separate aliquots: 0.3, 0.5 and 0.7 mL. Cells within the 0.3mL aliquot were blocked with anti-CD16/CD32 (1:200; BD Biosciences, San Jose, CA) for 20 min on ice. Suspensions were then split into two 150μl samples, and were either stained with anti—Ly6G-allophycocyanin (eBioscience) or isogenic tag and washed after 20 min incubation at 4°C. Data were collected on a BD FACSCalibur cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR). For protein lysate aliquots, 0.5mL BAL aliquots were treated with 10μL protease inhibitor cocktail set III, EDTA-free (Calbiochem, MA) and 20μl of 10% Triton X-100 (Sigma, MA) and mixed by inversion for 20 minutes at 4°C. Samples were used for mouse neutrophil elastase/ELA2 and MPO ELISA (R&D Systems, MN) according to the manufacturers protocol. The 0.7mL BAL aliquots were centrifuged (400 RCF, 5 min, 4°C), and cell-free supernatant was collected and used assay LDH and LTB₄ content as described.

Bronchial alveolar lavage (BAL) samples were obtained and processed as previously described^{26 (link)}. Lungs were flushed with 2 mL total (0.5 mL per wash, 4×) of HBSS supplemented with 0.3% FBS and 300 μM EDTA. Collected BAL was separated into three separate aliquots: 1.0, 0.5, and 0.5 mL. Cell count and viability measurements using trypan blue stain were performed using Countess Automated Cell Counter (Invitrogen). Cells were blocked with anti-CD16/CD32 (BD Biosciences, San Jose, CA) for 20 min on ice and processed for flow cytometry. For protein lysate samples, one of the 0.5 mL BAL aliquots was treated with 10 μL protease inhibitor cocktail set III, EDTA-free (Calbiochem, MA) and 20 μl of 10% Triton X-100 (Sigma, MA) and mixed by inversion for 20 minutes at 4 °C. Protein lysate samples were used for mouse neutrophil elastase/ELA2 and MPO ELISA (R&D Systems, MN) per manufacturers protocol. The other 0.5 mL BAL aliquots were centrifuged, and cell-free supernatant was collected and used for lactate dehydrogenase (LDH) release assay according to manufacturer’s instructions (Sigma).