Mouse il 12 p70 quantikine elisa kit

Supernatants from infected CT26WT cells with VSVΔ51 encoding Fluc or IL12 at MOI 0.1 were collected 24 hours post infection. The secretion of IL12 was measured using mouse IL-12 p70 Quantikine ELISA (Mouse IL-12 p70 Quantikine ELISA Kit, R&D system, Cat#M1270) following the manufacturer’s instructions. Absorbance values at 450nM were measured on a Multiskan Ascent Microplate Reader (MXT Lab System) and corrected for plate imperfections at 540. Data were analyzed using Prism.

Submandibular blood samples collected from mice were placed in BD Microtainer Serum Separator Tubes (BD Biosciences) and stored at room temperature for at least 30 min. The tubes were then spun at 5,000 RPM for 5 min, and the serum was collected and used in a Mouse IL-6 ELISA Ready-Set-Go! Kit, Mouse TNFα ELISA Ready-Set-Go! Kit (Thermo Fisher Scientific) and Mouse IL-12 p70 Quantikine ELISA Kit (R&D Systems) following the manufacturers’ instructions.

Mouse IL‐12p70 Quantikine ELISA Kit (M1270), Mouse IL‐10 Quantikine ELISA Kit (M1000B), Mouse IFN‐beta Quantikine ELISA Kit (MIFNB0) and Mouse IFN‐alpha ELISA Kit (42120‐1), Human IL‐10 Quantikine ELISA Kit (D1000B), Human IL‐12 p70 Quantikine ELISA Kit (D1200) were purchased from R&D Systems. Mouse IL‐6 ELISA Kit (70‐EK206/3‐96) and Mouse TNF‐a ELISA Kit (70‐EK282/3‐96) were purchased from Multisciences (Lianke) Biotech, Co., Ltd. The methods were performed according to the manufacturer's instructions.

For the ELISA assay (Human IL-12 p70 Quantikine ELISA Kit, R&D Systems, Minneapolis, MN, USA, or Mouse IL-12 p70 Quantikine ELISA Kit, R&D), the cell culture medium from the FaDu and CT26 cells was collected at different timepoints after GET designated in Figure 1. The cell culture medium was centrifuged and the supernatant was then divided to aliquots and stored at −80 °C. All the samples, standards and controls were assayed in duplicate. The ELISA assay was performed according to the manufacturer’s instructions. Within 30 min after the end of the assay, optical density (OD) for each well was measured using a spectrophotometer (Cytation 1, BioTek Instruments) at 450 and 570 nm. The reading value at 570 nm was subtracted from the 450 nm value to correct for optical imperfections in the plate. The duplicate readings for each standard, control and sample were averaged and then the average zero standard optical density (OD) was subtracted from them. The standard curve was created by reducing the data to generate a four-parameter logistic (4-PL) curve fit according to the manufacturer’s instructions. The obtained equation was then used to calculate the concentration of hIL-12 p70 or mIL-12 p70 in the samples. For each sample, the total concentration of the protein in the cell media was indicated as the average OD value × volume of cell media/number of cells.