Pge2 elisa kit

As previous studies have described⁴⁸ , CT-26 cells were seeded at 12-well plates for 12 h. Then, different concentrations of C-PC (0, 50, 100, 200, and 300 μg/mL) were added to culture media and incubated for 24 h. Subsequently, arachidonic acid was added to each well and after 1 h, the culture media were collected and cell derbies were removed by centrifuging. Prostaglandin E2 (PGE2) level in the cell-free culture medium was measured by employing PGE2 ELISA kits (Cayman Chemical Company, USA) according to the manufacturer’s instructions.

PBS was from Oxoid. DNAse- and RNAse-free water was from VWR. Recombinant human TNF and IL-6 ELISA Duoset were from R&D systems. Quantitect primer assays for TLR1-7 and 18S were from Qiagen. QuantiTect Reverse Transcription kit, RNeasy minikit, Leupeptin, pepstatin and LightCycler 480 SYBR Green I Master mix were from Roche Molecular Biochemicals. RNAlater was from Life technologies. FSL-1 and Pam₃CSK₄ were from Invivogen. [³H]-arachidonic acid ([³H]-AA), and liquid scintillation cocktail Ultima Gold were from NEN Perkin Elmer. AVX002 and Inhibitor 28 were provided by Avexxin AS (Trondheim, Norway). Arachidonyl trifluoromethyl ketone (AACOCF3, ATK) was from Enzo Life Sciences. Varespladib (LY315920) was from Selleckchem. CAY10502, CAY10590 and PGE₂ ELISA kit were from Cayman Chemicals. Phospho-cPLA2 (Ser505) antibody was from Cell Signal Technology. α-tubulin antibody was from Santa Cruz Biotechnology. Polyclonal goat anti-mouse immunoglobulins horse radish peroxidase-conjugated secondary antibody was from Dako. Hybond-C nitrocellulose membranes were from GE healthcare. NuPAGE gel system (10% Bis-Tris gel) was from Invitrogen. Hybond-C nitrocellulose membranes were from GE healthcare. SuperSignal West Femto Maximum Sensivity substrate was from Thermo Scientific. All other reagents were from Sigma-Aldrich.

Eight-week-old NC/Nga mice were purchased from RIKEN BioResource Center, Japan. Isoflurane was obtained from Piramal Healthcare Limited (Kuala Lumpur, Malaysia). DNFB, acetone, CS (MW, 70 kDa; deacetylation degree, 85%), HC (base form), and HT were purchased from Sigma Aldrich Chemicals Co. Ltd. (Kuala Lumpur, Malaysia). Halt protease inhibitor cocktail and cell lysis buffers were sourced from Thermo Scientific (Kuala Lumpur, Malaysia). The chemicals used to conduct immunological studies included IgE enzyme linked immunosorbent assay (ELISA) kit (Abcam Chemical, Malaysia), PGE₂ ELISA kit (Cayman Chemical, Malaysia), histamine ELISA kit (Abnova Chemicals, Malaysia), VEGF-α ELISA kit (Life Technologies, Malaysia), and multi-analyte profiling Procarta assay kit (Affymetrix, Malaysia). All other chemicals were of analytical grade and sourced from research laboratories of Universiti Kebangsaan Malaysia.

Fetal bovine serum (FBS), RPMI1640 medium, and PBS (Hyclon, Logen, UT); dairy cow peripheral blood neutrophil isolation kit (Hao Yang, Tian Jing, China); MH broth (Oxoid, Hampshire, UK); TLR2 receptor inhibitor (C29, MCE, China), TLR4 receptor inhibitor (TAK-242, MCE, China), NLRP3 receptor inhibitor (MCC950, MCE, China); COX-2 inhibitor (CAY10404), mPGES-1 inhibitor (MF63) (Cayman Chemical, MI, United States); gentamicin sulfate(Promega, Wisconsin, United States); Bovine IL-1β, IL-10, IL-8 ELISA Kits (Kingfisher, Biotech); Bovine IL-6 ELISA Kit (R&D Systems, California, United States); PGE₂ ELISA Kit (Cayman Chemical, MI, United States); M-PER mammalian protein extraction reagent, Halt protease inhibitor, pre-stained protein ladders, blocking buffer, starting block T20 (TBS), LIVE/DEAD Bac Light Bacterial Viability Kits (Thermo Fisher Scientific); SDS-PAGE loading buffer (Takara, Shiga, Japan); Pierce BCA Protein Assay Kit (Rockford, United States); SDS-PAGE gel electrophoresis kit (Solarbio, Beijing, China); Axy Prep Multisource Total mRNA Miniprep Kit (Axygen Scientific, Union City, United States); Primer Script RT Master Mix (Takara); SYBR Green Master (Rox) (Roche, Basel, Switzerland). All primers were synthesized by Generay (Shanghai, China).