Leupeptin hemisulfate

Lymphoprep™ gradient was purchased from Progen (Heidelberg, Germany). RPMI 1640 medium, fetal calf serum, trypsin 10x (25 mg/mL), trypsin-EDTA 10x (5 mg/mL trypsin and 2.2 mg/mL EDTA), and phosphate buffered saline (PBS, without Ca and Mg) were from PAA Laboratories Gmbh (Coelbe, Germany). L-Glutamine, penicillin, and streptomycin were from Invitrogen (Karlsruhe, Germany). Camptothecin was from Tocris (Eching, Germany); Triton-X 100, milk powder, and N,N,N′,N′-tetramethyl-1-,2-diaminomethane (Temed) were from Carl Roth (Karlsruhe, Germany); DMSO, lipopolysaccharide (LPS), phorbol, 12-myristate 13-acetate, CFSE, ammonium persulfate, bovine serum albumin, ethanol absolute, hydrochloric acid (37%), leupeptin hemisulfate, p-coumaric acid, pepstatin A, Ponceau S, trypan blue, Tween 20, and ionomycin were from Sigma-Aldrich (Taufkirchen, Germany).
Antibodies against p-38 (Thr180/Tyr182), p-ERK1/2 (Thr202/Tyr204), p-JNK, and p-c-Jun (Ser73) and the horseradish peroxidase- (HRP-) labelled secondary antibodies, anti-mouse and anti-rabbit, were from Cell Signaling Technology (Boston, USA); anti-human COX-2 was from R&D Systems (Wiesbaden, Germany); anti-human COX-1 and anti-human 5-LOX (mouse monoclonal, clone 33) were from Santa Cruz Biotechnology (Heidelberg, Germany); and mAb against β-actin was from Sigma-Aldrich (Taufkirchen, Germany).

Exosomes were isolated by step-wise ultracentrifugation as previously reported [11 (link),21 (link)] with a few modifications. Briefly, urine samples (≥30 mL) were collected by simple voiding and protease inhibitor (combination of 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF-HCl, Sigma-Aldrich, St. Louis, MO, USA), leupeptin-hemisulfate (Sigma-Aldrich, St. Louis, MO, USA), and NaN3 (Sigma-Aldrich, St. Louis, MO, USA)) were immediately added. To remove urinary sediments, the samples were centrifuged at 4000 rpm for 15 min at 4 °C and the resulting supernatants were collected and stored at −80 °C until exosome extraction. Frozen urine samples (15 mL) were thawed and vortexed for 1 min, centrifuged at 17,000 g for 15 min at room temperature, and the resulting supernatant was collected and ultracentrifuged at 200,000 g for 70 min at room temperature using a Beckman Coulter Optima L-80xp ultracentrifuge, rotor SW40Ti (Beckman Coulter; Brea, CA, USA). The resulting supernatant was discarded and the pellet was dissolved in 11 mL DPBS for washing, which was ultracentrifuged again at 200,000× g for 70 min at room temperature. The supernatant was discarded and the resulting pellet of exosomes was used for protein isolation or quantification of exosome particles.