Human tnf alpha duoset elisa

Serum samples (50 μl) were collected following centrifugation of blood samples at 1500 g for 10 min and stored at −20 °C. Biopsy media was collected, centrifuged and stored as described above prior to enzyme-linked immunosorbent assay (ELISA) tests. The levels of CD44 (CD44 Human ELISA Kit, Abcam, UK), OPN (Human OPN DuoSet ELISA R&D Systems, UK), TNF-α (Human TNF-alpha DuoSet ELISA, R&D Systems, UK) and IFN-γ (Human IFN-gamma DuoSet ELISA, R&D Systems, UK) were measured in serum and in biopsy media by ELISA following the manufacturer’s protocol. Samples were analysed in triplicate and data obtained for in vitro experiments is representative of four independent experiments.

TNFα and IFNγ levels were analyzed in the supernatants by using Human TNF-alpha DuoSet ELISA (DY008) or Human IFN-gamma DuoSet ELISA (DY285) from R&D systems according to the manufacturer’s protocol plus a TECAN^® Infinite F50^® microplate reader.

Plasma levels of TNF-α were assayed using the sandwich Human TNFalpha DuoSet ELISA (6 µl sample, standard range 600–6 pg/ml, intra-assay coefficient of variation [cv] of 2%, inter-assay cv of 23%, DY210, R&D Systems, Bio-Techne GmbH, Wiesbaden, Germany). Plasma levels of IL-6 were quantified by the sandwich Human IL-6 DuoSet ELISA (3 µl sample, standard range 1500–9 pg/ml, intra-assay cv of 1%, inter-assay cv of 7%, DY206, R&D Systems, Bio-Techne GmbH). Plasma levels of IL-10 were determined with the sandwich Human IL-10 DuoSet ELISA (100 µl sample, standard range 1000–4 pg/ml, intra-assay cv of 6%, inter-assay cv of 25%, DY217, R&D Systems, Bio-Techne GmbH). All of the samples from one stimulation were always run on the same plate.

The Nod2^+/+ and Nod2^-/- THP-1 monocytic cell line was cultured in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated FBS (Gibco), L-glutamine (Thermo Fisher), MEM non-essential amino acids (Thermo Fisher), sodium pyruvate (Thermo Fisher), HEPES (Thermo Fisher), and 0.05mM of 2-mercaptoethanol (Thermo Fisher). Cells were kept in culture at cell concentrations ranging from 2x10⁵ cells/mL to 8x10⁵ cells/mL and routinely verified negative for mycoplasma contamination by PCR analysis. THP-1 macrophages (PMA-Mac) were generated by adding PMA (5ng/ml) for 48h, followed by at least 2 days without PMA (35 (link), 67 (link)). PMA-Mac stimulation was performed in 96-well flat bottom plates at 1x10⁵ cells per well in a final volume of 200 μl. Cells were stimulated with two sequential treatments of 24 hours each. For the first 24 hours of treatment, cells were cultured in RPMI complete medium or with LPS at 1ug/mL, and washed to remove the stimuli. The second 24-hour treatment consisted of MDP at 10ug/mL or RPMI medium. Culture supernatants were collected after the second treatment and TNF-α levels were quantified by ELISA using the Human TNF-alpha DuoSet ELISA (R&D systems) following manufacturer recommendations.

Defective TNF-α production ex-vivo is a major trait of sepsis-induced immunosuppression (19 (link)), so this pro-inflammatory cytokine was evaluated as reference of PTX3 behavior. PTX3 and TNFα proteins from patients and HV in plasma and Truculture® supernatant were quantified using ELLA nanofluidic system (Biotechne, Minneapolis, MI, USA), according to the manufacturers' instructions. PTX3 and TNFα proteins after lysis of cellular pellets from untreated and cycloheximide conditions in the intracellular protein stability assay were also quantified using ELLA. Intracellular protein levels were expressed as a percentage of the maximal protein level.
These two proteins concentrations, from endotoxin tolerance model in PBMC and in THP1-Xblue™-MD2-CD14 cell culture supernatants, were detected using commercially-available ELISA kits Human TNF-alpha DuoSet ELISA and Human Pentraxin 3/TSG-14 DuoSet ELISA (R&D Systems, Biotechne), in accordance with the manufacturers' instructions. Results are expressed in pg/ml.

TNF level in supernatants of human macrophages was determined using human TNF-alpha DuoSet ELISA (DY210-05) (R&D Systems, Minneapolis, MN, USA), IFNβ level—using VeriKine-HSTM Human Interferon-Beta Serum ELISA Kit from PBL Assay Science (Piscataway, NJ, USA). Other cytokines (IL-12p70, IL-6, MCP-1, IL-8) were analyzed using BioPlex cytokine assays from Bio-Rad, in accordance with the instructions of the manufacturer, using the Bio-Plex Pro™ Reagent Kit III and Bio-Plex™ 200 System (Bio-Rad, Hercules, CA, USA).

The THP1 monocytic cell line was cultured in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated FBS (Gibco), L-glutamine (Thermo Fisher), Penicillin and Streptomycin (Thermo Fisher), MEM non-essential amino acids (Thermo Fisher), sodium pyruvate (Thermo Fisher), HEPES (Thermo Fisher) and 0.05mM of 2-mercaptoethanol (Thermo Fisher). Cells were kept in the culture at cell concentrations ranging from 2x10⁵ cells/mL to 8x10⁵ cells/mL and routinely verified negatively for mycoplasma contamination by PCR analysis. THP1 stimulation was performed in 96-well flat bottom plates at 1x10⁵ cells per well in a final volume of 200 μl. Cells were stimulated with two sequential treatments of 24 hours each. For the first 24 hours of treatment, cells were cultured in RPMI complete medium or RPMI medium with MDP at 100μg/ml. The second 24-hour treatment consisted of LPS (Invivogen) at 50ng/ml or RPMI medium. In selected experiments, the first treatment was MHY1485 (Sigma) at 2μM, rapamycin (Sigma) at 100nM, or wortmannin (Sigma) at 1μM. In some conditions, THP-1 macrophages were generated by adding PMA (5ng/ml) for 48h, followed by at least 2 days without PMA (121 (link), 122 (link)). Culture supernatants were collected after the second treatment and TNF-α levels were quantified by ELISA using the Human TNF-alpha DuoSet ELISA (R&D systems) following manufacturer recommendations.