Rat vegf quantikine elisa kit

The level of nerve growth factor (NGF), human growth factor (HGF), glial-derived neurotrophic factor (GDNF), and vascular endothelial growth factor (VEGF) in the medium of conditioned MSCs and in the conditioning media (M0CM, M1CM, Pol1, D10) were determined using absorbance-based sandwich ELISAs, following the manufacturers’ protocols. Samples were thawed at room temperature, vortexed, spun down at 1000 G for 20 min at 4 °C, and assayed using NGF beta rat ELISA Kit (#ERNGF, ThermoFisher Scientific, Waltham, MA, USA), Rat HGF ELISA kit (#MBS825055, MyBiosource Inc., San Diego, CA, USA), Rat GDNF PicoKineTM ELISA Kit (#EK0363, Boster Bio, Pleasanton, CA, USA), and Rat VEGF Quantikine ELISA Kit (#RRV00, R&D Systems, Bio-techne, Minneapolis, MN, USA). Absorbance readings were carried out in a FLUOStar Omega microplate reader (BMG Labtech, Ortenberg, Germany).

A colorimetric solid phase sandwich ELISA (Rat VEGF Quantikine ELISA Kit; R & D Systems, Wiesbaden-Nordenstadt, Germany) was used to determine VEGF concentrations in rat aqueous humor [33 (link), 34 (link)]. The assay was carried out according to the manufacturer’s instructions. Measurements were performed using a Microplate Reader (AESKU Reader; AESKU.DIAGNOSTICS, Wendelsheim, Germany).

Rat serum and liver tissue IL‐1β, IL‐18 (Rat IL‐1β, IL‐18 ELISA Kit; Invitrogen, Paisley, UK), histone H3 (Histone H3 ELISA kit; LSBio), and VEGF (Rat VEGF Quantikine ELISA Kit; R&D, Abingdon, UK) were measured with use of an ELISA. U937 medium IL‐1β and interferon (IFN)‐γ were assessed by ELISA (Human IL‐1β and IFN‐γ Elisa Kit; Invitrogen).

Frozen lung samples were homogenized in lysis buffer, and lysates were clarified by centrifugation at 13,000 g for 20 min at 4°C to remove cellular debris. The protein contents of the supernatants were quantitated by the Bradford method, using bovine serum albumin as the standard. Lung macrophage inflammatory protein-1 α (MIP-1α), tumor necrosis factor- α (TNF-α) and interleukin-6 (IL-6) levels were measured using the Milliplex MAP ELISA Kit according to the manufacturer’s protocol (Millipore, Billerica, MA, USA). Vascular endothelial growth factor (VEGF) levels were measured using the R & D Rat VEGF Quantikine ELISA kit according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA).

Specimens were isolated, cut into small fragments, homogenized, and incubated in cold lysis buffer (20 mM HEPES, 0.5 mM ethylene glycol bis (beta-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 1 mM dithiothreitol, and 0.32 M sucrose; pH 7.4) containing phenylmethanesulfonyl fluoride (protease inhibitors; LiankeBio; Hangzhou) for 30 min at 4 °C. Then they were centrifuged at 3000 rpm for 5 min at 4 °C. The supernatant was collected for examination. According to the manufacturers’ protocols, relevant active factors were quantified with the Soluble Collagen Assay Kit (Abcam, Cambridge, MA, US; ab241015), Rat Integrin-β1 ELISA Kit (Cusabio Biotech, Wuhan, China, code: CSB-E14205r), Rat E-Cadherin ELISA Kit (Abcam, Cambridge, MA, US; ab202413), Rat VEGF Quantikine ELISA Kit (R&D Systems Inc., Minneapolis, MN, USA; Catalog #:MFB00), Rat FGF2 Quantikine ELISA Kit (R&D Systems Inc., Minneapolis, MN, USA; Catalog #:MFB00), and Rat beta NGF ELISA Kit (Abcam, Cambridge, MA, US; ab193736). The signal was recorded by a microplate reader (Bio-Rad; USA), and the optical density measured at 450 nm (OD₄₅₀) was used to calculate the concentration of the protein of interest in the specimen.