Human ifn γ elisa development kit

Cell lysates from HDBEC cultured in gelatin-coated 12-well plates were prepared using a mixture of NuPAGE LDS Sample Buffer and NuPAGE Sample Reducing Agent (Thermofisher Scientific). Samples were loaded on NuPAGE Bis-Tris4%–12% protein gels. NuPAGE MOPS SDS Running Buffer supplemented with 200 μl of NuPAGE Antioxidant was used during electrophoresis, and the gels were transferred using NuPAGE Transfer Buffer. SeeBlue Pl2 USD Pre-Stained Standard (ThermoFisher Scientific) was used as a loading marker. Proteins were blotted onto an Amersham nitrocellulose blotting membrane 0,2 μm-0,45 μm and Amersham ECL prime was used as a detection reagent (GE Healthcare Sciences). Primary antibodies were anti–IDO (D5J4E) Rabbit mAb (Cell Signaling Technology) and Mouse Anti-β-Catenin (Clone 14/Beta-Catenin, BD Biosciences). Horseradish peroxidase-labeled secondary antibodies (GE Healthcare and Sigma) were used. For detection of hIFNγ in isolated T cells from PBMCs from healthy donors, the human IFNγ ELISA development kit (MabTech) was used.

Urethral mucosa tissues were processed as described^{74 (link)} to obtain tissue lysates. The cytokines/chemokines CXCL4/PF4, IL-13, M-CSF, MCP-1/CCL2, TRAIL, RANTES/CCL5, MIP-1α/CCL3, GM-CSF, IL-4, MIP-3α/CCL20, and MCP-4/CCL13 were quantified using the Luminex technology (LXSAHM, R&D) on a Bio-Plex 200 (Bio-Rad) according to the manufacturer’s recommendations. IFN-γ was quantified using the Human IFN-γ ELISA Development Kit (Mabtech), according to the manufacturer’s instructions. The lower limit of quantification of each cytokine/chemokine is 2 pg/mL (CCL13/MCP-4 and IFNγ), 3 pg/mL (CCL20/MIP-3α), 4 pg/mL (GM-CSF), 7 pg/mL (CCL5/RANTES), 10 pg/mL (TRAIL and CCL2/MCP-1), 16 pg/mL (IL-4), 100 pg/mL (CXCL4/PF4 and CCL3/MIP-1a), and 140 pg/mL (IL-13 and M-CSF). Correlograms were generated in R using cor, cor_test_mat, and corrplot functions.
S100A8/S100A8 heterodimer was measured by Quantikine ELISA kit (DS8900, R&D) according to the manufacturer’s recommendations.
Cytokine levels were normalized by the protein amount in tissue lysates (mg/mL), quantified by NanoDrop spectrophotometer (ThermoFisher Scientific) using the program Protein A280.

PBMCs were isolated by density centrifugation with Ficoll-Paque (GE Healthcare, Chicago, IL, USA) from healthy donor buffy coats acquired from the blood bank at Uppsala University hospital. PBMCs were cultured alone or co-cultured either with L363 or U266-84 cells in a ratio of 2:1 at a concentration of 1 × 10⁶ cells/mL in 6-well plates (3 × 10⁶ cells in total). After 24 h, cells were infected with the respective virus by direct addition of virus into the respective well (100 FFU/cell). PBMCs cultured alone and one co-culture well were left uninfected. Forty-eight hours of post infection, cells were harvested and cell culture supernatants were taken for IFN-γ detection (Human IFN-γ ELISA development kit, Mabtech AB, Nacka Strand, Sweden). Cells were stained for flow cytometry analysis as described above with the following antibodies purchased from BD Biosciences: CD45 (FITC; clone 2D1), CD16 (PE; clone B73.1), CD56 (PE; clone My31), CD3 (PerCP; clone SK7), CD4 (PE-Cy7, APC-H7; clone SK3), CD8 (APC; SK1), CCR7 (FITC; clone 150503), CD45RA (APC-H7; clone HI100), PD-1 (PE; clone EH12.1), CD69 (FITC; clone L78), CD25 (PE, clone 2A3), CD127 (PE-Cy7; HIL-7R-M21). CD3 (PerCP; clone UCHT1), CD8a (FITC; clone RPA-T8), and CD107a (PE; clone H4A3) were purchased from BioLegend.