Human pmn elastase elisa kit

Deionized water Milli-Q (conductivity < 0.1 μS cm⁻¹) (Millipore system, Molsheim, France) and analytical grade chemicals were used without further purification unless otherwise stated. PSU in pellets with average molecular weight (MW) 35,000 Da (CAS Number 25135-51-7); 1-methyl-2-pyrrolidone (NMP) (CAS Number 872-50-4); Polyvinylpyrrolidone (PVP) K30 (CAS Number 9003-39-8); HEPES sodium salt (CAS Number 75277-39-3); phorbol-12-myristate-13-acetate (PMA) (CAS Number 16561-29-8); acetonitrile (CAS Number 75-05-8); Elastase, Human Neutrophil (CAS 9004-06-2); Elastase Substrate V, Fluorogenic (CAS 72252-90-5); and single-side polished N-type silicon wafer (CAS Number 7440-21-3) were all purchased from Sigma-Aldrich Co., Saint Louis, MO, USA. Sivelestat, neutrophil elastase inhibitor (CAS Number 127373-66-4), and Human PMN Elastase ELISA Kit were acquired from Abcam, Cambridge, UK. Dimethyl sulfoxide (DMSO) (CAS Number 127373-66-4) was purchased from VWR Chemicals, Radnor, PA, USA. Monoclonal antibodies CD42a (GRP-P), PE, and CD62P (P-Selectin), and APC, used for the determination of platelet activation, were purchased from Invitrogen, ThermoFisher Scientific, Waltham, MA, USA.

Platelets, neutrophils, and lymphocyte were measured routinely in the clinical laboratory department of our hospital. NLR and PLR were calculated by dividing absolute neutrophil count and platelets count by the absolute lymphocyte count, respectively. Tumor biomarker CA19-9 was also measured in the clinical laboratory department of our hospital and a cut-off value of 45,500 U/mL, previously determined in our research [15 (link)], was used.
The analysis of cell-free DNA (cfDNA) was performed as previously described [15 (link)]. In brief, plasma was obtained from 10 mL of peripheral blood before treatment and cfDNA was extracted from 3 mL of plasma with the QIAamp Circulating Nucleic Acid Kit and the vacuum system QIAvac 24 Plus (Qiagen, Hilden, Germany). The Quantus fluorometer (Promega, Madison, WI, USA) and the Agilent 2200 TapeStation system (Agilent, Santa Clara, CA, USA) with the High Sensitivity D1000 ScreenTape assay were used for measuring cfDNA concentration and fragmentation, respectively. The OncoBEAM™ RAS assay (Sysmex Inostics GmbH, Hamburg, Germany) was used for the analysis of RAS mutations in cfDNA and the determination of RAS mutant allelic fraction (MAF) in plasma.
For the quantification of elastase circulating levels, the Human PMN Elastase ELISA Kit (Abcam, Cambridge, UK) was used following the manufacturer’s instructions.

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.