Phenyl methyl sulfonyl fluoride (pmsf)

For in vivo studies, PMN or BMDN media was concentrated using a 3kDa cutoff centricon (Millipore) and 50μg/100μL was intratumorally injected. For boiling, PMN media was boiled at 95^oC for 5min and centrifuged at 15,000xg for 10min to remove denautred proteins. For dialysis, PMN media was placed into a Slide-a-Lyzer™ cassette (3.5kDa cutoff, ThermoFisher Scientific) and dialyzed against 2×4L of PBS at 4^oC for 4h. For size exclusion, PMN media was filtered through a 3kDa cutoff centricon (Millipore). For PMSF-inactivation, PMN media was treated with PMSF (1mM, Sigma) or A1AT (42nM, Athens Research & Technology) and incubated at room temperature for up to 2h. Residual PMSF was eliminated with a PD-10 desalting column (GE Healthcare Life). Inhibition of ELANE catalytic activity was confirmed using a chromogenic substrate activity assay (see below). For serum-spiking experiments, human, mouse, or fetal bovine serum (1% or 10%) was added to PMN media prior to or after exposing cancer cells. For immunodepletion studies, ELANE or ECP were immunoprecipitated using anti-ELANE (N2C3, GeneTx) or anti-ECP (MBS2535165, MyBioSource) antibodies coupled to Pierce™ Protein A/G Magnetic Beads (ThermoFisher Scientific).

Catalytic activity was measured using the chromogenic substrate N-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma, 100μg/mL) according to the manufacturer’s protocol. Absorbance was measured at 405nm using an accuSkan GO UV/Vis microplate spectrophotometer (ThermoFisher Scientific). 1 unit is defined as the amount of enzyme hydrolyzes 1nmol of substrate/min at 37^oC. For inactivation, ELANE or PPE was incubated with PMSF (1mM, Sigma) or A1AT (at indicated dose) for 2h. Residual PMSF was eliminated with a PD-10 desalting column (GE Healthcare Life). To monitor the effects of ECP on ELANE activity, ELANE (10nM) was incubated with various doses of ECP (0–180nM) at various substrate concentrations (0–1.7mM).