Phenylmethylsulfonyl fluoride pmsf

Levels of cytochrome C in the cell cytoplasm of A549 cells forming ALI multilayered mono-cultures were quantified by Enzyme ImmunoSorbent Assay (ELISA) (Cytochrome c ELISA Kit, Invitrogen, Biosciences Ltd, Ireland), following the manufacture’s protocol. ALI MCCs were exposed for 72 h to docetaxel, vinblastine, cytarabine and methotrexate. Drugs were tested at their nominal IC₅₀ concentration, as for GDSC database, and were added to the cultures by direct inoculation. Untreated cultures were also tested as negative control (NT). Detection of cytochrome c released from the mitochondria to the cytosol was achieved by selective lysis of the cell membrane, using a Cell Extraction Buffer (Invitrogen, Biosciences Ltd, Ireland), supplemented with protease inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF) (both from Santa Cruz Biotechnology Inc., Fannin Limited, Dublin, Ireland). For assay read-outs, the optical density of each well at λ = 450 nm was determined using an Epoch microplate reader.

Histopaque, p-nitrophenyl valerate (pNPVa), and LPS (E. coli 055:B5) were purchased from Sigma (St. Louis, MO, USA). AIM V^® Serum Free Medium was purchased from Thermo-Fisher (Waltham, MA, USA). RIPA lysis buffer and protease inhibitors (phenylmethylsulfonyl fluoride, PMSF; 4-(2-aminoethyl)benzenesulfonyl fluoride, AEBSF; bestatin; pepstatin A; leupeptin hemisulfate; and aprotinin) were from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies used for Western blots (anti-CES1, anti-MAGL, β-actin, goat anti-rabbit, and goat anti-mouse) were purchased from Abcam (Cambridge, MA, USA). Authentic 2-AG, anandamide, AA, and its deuterated analog AA-d8 were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Small molecules JZL184 and WWL113 were purchased from Sigma. CpG was purchased from InvivoGen (San Diego, CA, USA). Primary CES1 and MAGL antibodies for flow cytometry were purchased from Abcam. Fluorescence secondary antibodies for flow cytometry and antibodies against IL-6 used to neutralize IL-6 or perform ELISA were purchased from Biolegend (San Diego, CA, USA). For some of the experiments, monocyte-depleted (n = 1) and whole PBMCs (n = 5) were purchased from Astarte Biologics (Bothell, WA, USA).

To determine the impact of RZ on the hippocampal BDNF, mice receiving ADR or RZ were euthanized for 4 weeks after the initiation of RZ treatment, and BDNF ELISA was performed as described [34 (link)]. Brains were immediately extracted from the skull (N = 8 to 9 mice per group). The hippocampus was microdissected from each cerebral hemisphere, flash-frozen by immersing the cryo-vials in the liquid nitrogen, and stored at − 80 °C until assayed. Each hippocampus was weighed and transferred into 500 μl ice-cold lysis buffer (NPER, Neuronal Protein Extraction Reagent, ThermoScientific) containing sodium orthovanadate (0.5 mM, Santa Cruz), phenyl-methylsulfonyl fluoride (PMSF, 1 mM, Santa Cruz), aprotinin (10 μg/ml, Santa Cruz), and leupeptin (1 μg/ml; Santa Cruz). Tissues were then sonicated individually and centrifuged at 4 °C, and the supernatants were collected and diluted at 1:5 or 1:10 with ice-cold Dulbecco’s PBS (Gibco). The supernatants were acidified to pH 2.6 and then neutralized to pH 7.6. The BDNF levels were assayed using a commercially available ELISA kit (E-EL-M0203, Elabscience Biotechnology) and uncoated ELISA plates (Nunc MaxiSorp, Biolegend). The colorimetric measurements were performed at 450 nm wavelength using a microplate reader (BioTek SynergyMx).