Human il 8 elisa kit

The PMA was purchased from Sigma-Aldrich (St. Louis, MO, USA). The SARS-CoV-2 Spike S1 subunit protein (synonyms: Spike protein, S Protein, S1 Subunit, host cell receptor-binding domain (RBD)) was from RayBiotech (Peachtree Corners, GA, USA). The human IL-8 ELISA kit was procured from R&D Systems (Minneapolis, MN, USA). The culture media DMEM was purchased from Corning Mediatech, Inc. (Manassas, VA, USA), and F-12K was from GE Healthcare Biosciences (Marlborough, MA, USA). The fetal bovine serum (FBS) was from Corning (Corning, NY, USA), antibiotic–antimycotic was from Gibco (Gaithersburg, MD, USA), and penicillin–streptomycin was purchased from Hyclone (Logan, UT, USA). Human nasopharyngeal carcinoma KBP cells were grown in DMEM high glucose media, and non-small cell lung cancer A549 cells were grown in F-12K media supplemented with 10% FBS and antibiotic/antimycotic, as previously described [30 (link),31 (link)].

Culture media of apical and basolateral compartments were collected at 24 h post infection, centrifuged and supernatant was stored at − 80 ℃ until the analysis was carried out. Selection of cytokines for detection using ELISA methods was guided by significant upregulation of gene expression based on RT-qPCR analysis. Commercially available sandwich ELISA kits, namely bovine TNFA ELISA kit (Thermo Scientific) and human IL8 ELISA kit (R&D Systems), were used as reported previously^{62 (link),63 (link)}. The measurement was obtained with SpectraMax i3x microplate reader (Molecular Devices) according to the manufacture’s protocol. The assays were performed in duplicate from three biological replicates with two technical replicates per experiment.

The secretory activity of the ASCs in each scaffold sample was assessed by means of the levels of VEGF and IL-8 accumulated in the conditioned medium during scaffold cultivation. Fragments of scaffolds before and after cryopreservation were cultured in the wells of a culture tablet, each fragment in 2 mL of medium. Sampling was carried out within the control periods: 3 days after separation into fragments, 24 h after defrosting, 96 h after defrosting. The selected aliquots were frozen and stored at −40 °C for no more than 2 months. The amounts of VEGF and IL-8 in the medium were measured using enzyme immunoassay (EIA). The analysis was carried out in accordance with the manufacturer’s protocol. The amount of detectable protein of each type was measured in pg/mL in accordance with calibration curves constructed from the optical densities of VEGF standards using the VEGF-ELISA-BEST kit (cat. DVE00, R&D Systems, Bio-Techne^®, Minneapolis, Minnesota, USA) and the IL-8 standards from the Human IL-8 ELISA Kit (cat. BMS204-3 Thermo Fisher Scientific, Waltham, MA, USA). When setting up the EIA, the optical density was measured using an INFINITE F50 microplate reader (Tecan Austria GmbH, Grödig, Austria) at 450 nm. Measurements were run according to the manufacturer’s protocol.

Post E. coli infection, HT-29-MTX cell supernatant was collected and analyzed for IL-8 using a Human IL-8 ELISA Kit (R&D Systems, QuantakineR) according to the manufacturer’s directions. The intra-assay coefficient of variation (CV) and the inter-assay CV were <5%.

Total IgE and anti-HDM IgE levels in the serum were measured using BD OptEIA™ ELISA kits (555248, BD Biosciences, Franklin Lakes, New Jersey, USA) and mouse anti-HDM IgE ELISA kit (3037, Chondrex, Woodinville, Washington, USA), following the manufacturer’s instructions. The standard and duplicated samples were diluted 3000 times for total IgE detection and 25 times for anti-HDM IgE detection with diluted buffer, respectively. To measure IL-6 and IL-8 release levels in A549 cells, the human IL-6 ELISA kit or human IL-8 ELISA kit (R&D Systems, Minneapolis, Minnesota, USA) were used following the manufacturer’s protocols.

The RNA extraction kit was purchased from Qiagen Sciences (Germantown, MD, USA). The SuperScript™ FirstStrand cDNA Synthesis kit, and SYBR green PCR reagent were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The PAF-R and GAPDH primers were purchased from SABiosciences (Valencia, CA, USA). The PAF-R antagonist, WEB2086, CPAF, and imipramine were purchased from Cayman Chemicals Co. (Ann Arbor, MI, USA). The human IL-8 ELISA kit was from R&D Systems (Minneapolis, MN). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).