Human trail tnfsf10 quantikine elisa kit

sfTRAIL protein contained in filtered supernatants were quantified by ELISA, using Human TRAIL/TNFSF10 Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s instructions. Serially diluted rhTRAIL (R&D Systems, Minneapolis, MN, USA) was used for indirect quantification. It is worth to note that along the experiments, sfTRAIL protein was produced in two opportunities. Protein quantification was performed by ELISA at each sfTRAIL production.

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.