Human tnf α elisa kit

ADMECs were seeded onto different matrices previously cut into small pieces with a biopsy punch under sterile conditions, or grown to confluence in 24-well plates (BD Falcon) in serum-free medium and stimulated for 24 h with interferon (IFN)-γ (100 U/mL), tumor necrosis factor (TNF)-α (100 ng/mL) or interleukin (IL)-1β (10 ng/mL) (all purchased from Peprotech, Milan, Italy). Cells seeded onto matrices were cultured for 36 h at 37 °C in a humidified atmosphere in a 5% v/v CO₂ incubator; the supernatant was then collected. The levels of placental growth factor (PlGF), VEGF-A, TNF-α, and IL-8 were determined with a commercial ELISA kit following the manufacturer’s protocol (Human PlGF ELISA kit, Human ANGPT-1 ELISA kit and Human VEGF-A ELISA kit, Sigma Aldrich; Human TNF-α ELISA KIT, Diaclone SAS; IL-8 Human ELISA Kit, Invitrogen Thermo Fisher Scientific).
The initial quantitative determination of IL-8/CXCL8, monocyte chemoattractant protein (MCP)-1/CCL2, macrophage inflammatory protein (MIP)-1α/CCL3, regulated upon activation, normal T cell expressed and secreted (RANTES)/CCL5, and IL-6 was performed by a bead-based multiplex immunoassay (Biorad) and a Bioplex 200 system (Biorad Laboratories, Hercules, CA, USA), as previously described [32 (link)].

Caco-2 cells (2 × 10⁵) were seeded into six-well plates and grown in complete medium for 14 days. Then, the medium was replaced by DMEM/F12, and the cells were treated with the two peptides (0.05 mg/mL) for 24 h. Inflammation stress was induced using 0.01 mg/mL LPS. The TNF-α concentration in cell supernatants was determined employing a human TNF-α ELISA kit according to the instructions of the manufacturer (Diaclone SAS, Besancon Cedex, France). Briefly, 100 μL of cell supernatant and TNF-α standard were added in duplicate to the wells. Samples were incubated for 3 h at room temperature with biotinylated anti-TNF-α at room temperature. The plate was washed three times with 300 µL of washing buffer. Then, 100 μL of streptavidin–HRP was added into the wells, and the plate was incubated for 30 min at room temperature and then washed three times. At this point, 100 µL of ready-to-use 3,3′,5,5′-tetramethylbenzidine substrate solution was added to each well and incubated for 15 min at room temperature. At the end, 100 µL of H₂SO₄ (stop reagent) was added, and the absorbance was detected at 450 nm using a plate reader (TECAN Infinite^®® M200 PRO, Männedorf, Switzerland).

Immunoglobulin A (IgA, g/L) was determined with the immunoturbidimetric method and the diagnostic kit IGA 2073755 322 using the Cobas Integra 400 plus biochemical analyzer (Roche Diagnostics, Basel, Switzerland), and immunoglobulin G (IgG, g/L) was determined with the IGGT kit (turbidimetric method). C-reactive protein (CRP, mg/dL) was determined by immunoturbidimetric method with the reinforcement of latex particles, using the diagnostic kit C-Reactive Protein Latex (CRPLX) and the Cobas Integra 400 plus biochemical analyzer; the precipitate was measured at the turbidimetric wavelength of 552 nm (Roche Diagnostics, Basel, Switzerland). The concentration of interleukin-6 (IL-6, pg/mL) was measured with the Human IL-6 High Sensitivity ELISA KIT Cat diagnostic kit (No. 950.035.096, Diaclone SAS, Besancon, France). Tumor Necrosis Factor α (TNF-α, pg/mL) was measured with the Human TNF α ELISA KIT (Catalog No. 1x90 950.090.096, Diaclone SAS, Besancon, France).

Differentiated THP-1 macrophages (see above) were pre-treated with either 200 nM solutions of the tested complexes, or 200 nM solution of prednisone dissolved in 0.1% DMF or the vehicle (0.1% (v/v) DMF solution) for 1 h, as described previously [54 (link),59 (link)]. The concentration of TNF-α was measured using a Human TNF-α ELISA Kit (Diaclone, Besançon, France), according to the manufacturer’s manual. Each experiment was conducted in triplicate.

Cells were exposed to either normoxia or IH for 24 h, culture medium was collected, and the concentration of RETN, TNFα, and CCL2 was measured by using a Human Resistin (RETN) ELISA kit (R&D Systems, Minneapolis, MN), Human TNFα ELISA kit (Diaclone SAS, Besançon, France) and Human C-C motif chemokine ligand 2 (CCL2) ELISA kit (R&D Systems) according to the instructions of the suppliers.

The ACE2 activity was measured as previously described [16 (link)] using fluorogenic substrate 7-methoxycoumarin-4-yl) acetyl-Ala-Pro-Lys(2,4-dinitrophenyl)-OH (Mca-APK(Dnp)) Mca-Ala-Pro-Lys(Dnp)-OH (BioVision Inc., CA, USA). Briefly, plasma was diluted 1:10 in ACE2 reaction buffer containing protease inhibitors (10 μM Bestatin-hydrochloride, 10 μM Z-prolyl-prolinal, (Sigma, MO, USA), 5 μM Amastatin-hydrochloride, 10 μM Captopril in a buffer of 500 mM NaCI, 100 μM ZnCI2, and 75 mM TRIS HCI, pH 6.5). All chemicals were from Santa Cruz (CA, USA) if not stated otherwise. The reaction was performed at 37 °C in black 96-well microtiter plates in a total volume of 200 μL using a fluorescence plate reader (TECAN^® infinite 200 PRO) at an excitation wavelength of 320 nm and emission wavelength of 405 nm. Enzymatic activity was determined from a fluorescence rate increase over a 10–120 min time course, and the activity was reported as relative fluorescence units (RFU)/min.
The TNF-alpha evaluation was performed on plasma samples according to the manufacturer’s instruction (Diaclone, Human TNF-α ELISA Kit; #950.090.192).
The measurements of Sirt1 and ACE2 activities and TNF-alpha levels were performed in a blinded fashion. All data are expressed as the mean ± SD of three independent experiments.

Differentiated THP1-Blue™ NF-κB macrophages (see the chapter 3.6) were pre-treated for 1 h with 2 µM solutions of the tested compounds 8, 12, 6, 17, cinnamic acid, and prednisone dissolved in DMSO, or only the vehicle [0.1% (v/v) DMF solution]. The inflammatory-like response was triggered by adding 1 μg/mL of LPS from E. coli 0111:B4 (Sigma-Aldrich) to the pre-treated macrophages; the control cells were left without LPS treatment. After 24 h, the supernatants were collected and the concentration of TNF-α was measured using a Human TNF-α ELISA Kit (Diaclone, Besançon, France), according to the manufacturer’s manual.