Human tnf α quantikine elisa kit

HT-29 cells were seeded (3.5 × 10⁴ cells/well) on transwell culture inserts [(Transparent PTFE membrane coated collagen, 0.4 μm pore size) Transwell-COL, Corning Incorporated, NY, USA], and cultured at 37°C under 5% CO₂ for 5–6 days until confluence was reached (TEER value ~601 Ω cm²). This setup was put in 6 well cell culture plates for co-culture with PBMCs that were seeded in the lower basolateral chamber. To analyze the anti-inflammatory activity of LABs in the co-culture setup, apical monolayers of HT-29 cells were stimulated with L. brevis, L. curvatus and L. pentosus (5 × 10⁷ cells/ml) for 48 h, then with Salmonella for 12 h. The CFS were collected from both apical and basolateral sides and stored at -4°C until estimation of the protein level of TNF-α using a commercially available enzyme-linked immunosorbent assay kit (Human TNF-α Quantikine ELISA kit, R & D system, MN, USA) according to the manufacturer’s instructions. In addition, the expression of IL-10 and TFG-β in HT-29 cells was analyzed by RT-PCR.

The Human TNF‐α Quantikine ELISA kit and Human Serpin INF‐γ Quantikine ELISA kit awere purchased from R&D Systems Inc., to detect the concentrations of TNF‐α and INF‐γ in the CM, according to the manufacturer's instructions. The absorbance at 450 nm was measured using a microplate reader and 540 nm was set as the reference wavelength.

Human monocytic cells U937 were differentiated towards macrophages following a previously published protocol [27 (link)]. Briefly, 5 × 10⁵ cells/mL were cultured in RPMI-1640 with 10% heat-inactivated FBS and 100 ng/mL PMA (phorbol-12 myristate 13-acetate, Sigma Aldrich, St. Louis, MO, USA) for 48 h, and then the culture medium was changed. After 24 h, the cells were gently scraped and treated with 100 ng/mL LPS (Sigma Aldrich, St. Louis, MO, USA) for 10 min. The cells (un-stimulated and stimulated) were then seeded in 24-well plates and incubated with exosomes or vehicle (control) for 18 h, after which the medium was collected and the TNFα concentration was quantified using a human TNFα Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA).

To quantify mRNA levels, quantitative real-time PCR was carried out using TaqMan Probe and Premix Ex Taq (Probe qPCR; TaKaRa Bio Inc., Shiga, Japan) in a TaKaRa PCR Thermal Cycler Dice TP960 (TaKaRa Bio) according to the manufacturer’s protocols. To normalize mRNA expression levels, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous internal control. Primers and probes used for RT-PCR are described in Table 2.
ELISA (Enzyme-linked immuno-sorbent assay) HAEC and THP-1 cells were treated with PAPC, OxPAPC or H₂OxPAPC for 22 h. The IL-8 (HAEC) and TNF-α (THP-1) contents in the culture media were determined using Human CXCL8/IL-8 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) and Human TNF-α Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA), respectively, according to the manufacturer’s protocol.

PMA-differentiated THP-1 cells were stimulated with the DP-1 supernatant with or without pretreatment with PKR inhibitors (2-AP and C16) or luteolin for 24 h. The expression levels of TNF-α were measured using the Human TNF-α Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

We collected peripheral blood samples at the beginning of hemodialysis during the first session of the week. Hemoglobin level, white blood cell counts, blood biochemistry including blood urea nitrogen (BUN), creatinine, albumin, total cholesterol, triglyceride, calcium, and phosphate were measured. Kt/V and normalized protein catabolic rate (nPCR) were calculated to represent dialysis adequacy and dietary protein intake; high sensitivity C-reactive protein (hsCRP) nephelometry (from Siemens) and intact-parathyroid hormone (i-PTH) immunoradiometric assay (from CISBio International) were assessed at the same time. Peripheral blood mononuclear cells (PBMCs) were isolated and submitted for flow cytometry analysis. LRG1 was measured by ELISA (from RayBio Human assay). The plasma levels of inflammatory cytokines, TNF-α and IL-6, were assayed with human TNF-α Quantikine ELISA kit and human IL-6 Quantikine HS ELISA kit and, respectively (from R&D systems).