Human tnf α ultrasensitive elisa kit

Saliva collection and processing were done as published [31 (link)]. Salivary stimulation was achieved by chewing unflavored chew paraffin wax pellets (Glee Gum, Verve Inc., Providence, RI). Saliva was stored at −80°C until cytokine assays were performed. Salivary interleukin-1 (IL-1β) and tumor necrosis factor-α (TNF-α) levels were assessed using human IL-1β ELISA kit (Invitrogen, Thermo Fisher Scientific, CA, USA) and Human TNF-α Ultrasensitive ELISA kit (Invitrogen, Thermo Fisher Scientific, CA, USA) using the manufacturer’s protocol. The absorbance was read spectrophotometrically at 450 nm using a GloMax Discover instrument v3.0. (Promega Corp, WI, USA). Using the standard equation curves, saliva IL-1β and TNF-α concentrations were determined.

TNFα and TRAIL mRNA levels were determined by quantitative real-time PCR (qRT-PCR) analysis. Total RNA extraction and cDNA synthesis were performed as previously described [28 (link)]. TNFα and TRAIL mRNA levels were assessed by Taqman Gene Expression Assay purchased from Life Technologies (TNFα: Hs01113624_g1, TRAIL: Hs00921974_m1) and the levels of 28S rRNA by SYBR®Green qPCR assay from Applied Biosystems (Darmstadt, Germany) according to the manufacturer's instructions using the 7900HT fast real-time PCR system from Applied Biosystems (Darmstadt, Germany); 28S rRNA forward primer: TTGAAAATCCGGGGGAGAG; reverse primer: ACATTGTTCCAACATGCCAG. The relative expression of the target gene transcript and reference gene transcript was calculated as ΔΔC_t. 28S rRNA was used as reference gene. TRAIL protein levels in whole cell lysates were determined by Human TRAIL/TNFSF10 Quantikine ELISA Kit obtained from R&D Systems (Wiesbaden, Germany) and TNFα levels in the supernatant of cells were determined by Human TNFα Ultrasensitive ELISA Kit purchased from Invitrogen (Carlsbad, USA) according to the manufacturer's instructions.