Human cytokine 30 plex kit

The spleens of mice were collected at 7 days after the third immunization and prepared into spleen cell suspensions. Three mice per group were included. In total, 2.5 × 10⁶ spleen cells was added to a 12-well cell-culture plate. In total, 10 μg/mL CDV-F or CDV-H protein, which was a stimulant, was also added to the plates. The wells, to which were added ConA, were positive controls, and those to which the stimulant was not added were negative controls. The plates were incubated in a 5% CO₂ incubator at 37 °C for 60 h. IFN-γ, IL-2, TNF-α, IL-4, IL-6 and IL-10 in the supernatant were evaluated by V-PLEX Human Cytokine 30-Plex Kit with measurements by Meso QuickPlex SQ120 (Meso Scale Discovery, Rockville, MD, USA).

The MSD assay detection kits were used to evaluate the inflammatory factors in the tears of the patients. Cytokine secretion was evaluated by V-PLEX Human Cytokine 30-Plex Kit with measurements by Meso QuickPlex SQ120 (Meso Scale Discovery, Rockville, MD, USA). After drawing the standard curve, the antibody to be tested was diluted. According to the experimental configuration, a 50 ul tear sample (the time on suture loosening and the last follow-up) was added to the corresponding hole, and the antibody was detected, and shaken at room temperature for 2 h. The MSD plate (Meso Scale Discovery, Rockville, MD, USA) was cleaned, and 25ul antibody was added to each hole and shaken at room temperature for 2 h. Clean the MSD board, add 150ul of prepared board reading liquid to each hole, and check it on the Meso QuickPlex SQ120.

Cell-free urines were analyzed on a SECTOR Imager 6000 (Meso Scale Discovery) using the V-PLEX Human Cytokine 30-Plex Kit to measure levels of IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-16, IL-17A, IFN-γ, TNF-α, TNF-β, GM-CSF, VEGF, CCL2, CCL3, CCL4, CCL11, CCL13, CCL17, CCL22, CCL26, CXCL8 and CXCL10, and using an ultrasensitive single-plex assays for sIL-6R. Conventional ELISA kits were used to measure creatinine, cystatin C, HSA, MMP8, MMP9 and RBP4 (R&D Systems) as well as fibrinogen (Abcam). HNE was measured using a B.I.T.S. ELISA kit (Mologic); activated PGP, desmosine, FMLP and NGAL were measured using validated in-house developed ELISA kits (Mologic).