Mouse vegf elisa kit

Based on the fourfold hydrogen bonding supramolecular ureido-pyrimidinone units were coupled with alkyl-urea to 10k poly(ethylene glycol) to synthesize the UPy-hydrogel (SyMO-Chem BV, Eindhoven, The Netherlands)^{14 (link)}. A 10 wt% UPy-hydrogel was made by dissolving 10 wt% UPy-PEG in 90 wt% saline to a final pH 9. After one hour of stirring, the liquefied UPy-hydrogel was mixed with mouse recombinant VEGF and IGF1 (V4512–5UG and I8779–50UG, Sigma-Aldrich, St Louis, MO, USA) to a concentration of 500 ng/ml of both GF. In addition, a second batch of the UPy^GF-hydrogel was mixed with 13,6 μg/ml USPIOS (Sinerem, Guerbet, Villepoint, France) for in vivo tracking after delivery. To assess the cumulative release of IGF1 and VEGF, 100 μl of the liquefied UPy-hydrogel was placed in a pipette at 24-well plate millicell hanging culture inserts (PIEP12R48, polyethylene terephtalate, 8.0μm; Merck Millipore, Darmstadt, Germany) and solidified by decreasing to pH 6.5 while adding hydrochloric acid. The MilliPores where surrounded with 800 μl phosphate-buffers saline (PBS) and placed at a rotational shaker (100 rpm) at 37 °C for 7 days. The PBS surrounding the inserts was refreshed daily and the collected supernatant was quantified for VEGF and IGF1 by enzyme-linked immunosorbent assay detection (Mouse IGF1 ELISA Kit and Mouse VEGF ELISA Kit, Sigma-Aldrich, St Louis, USA).

The effect of different treatments on vascular endothelial growth factor (VEGF) expression was measured using mouse VEGF ELISA kit (Sigma). EMT6/P cells were cultured at a concentration of (1.5 × 10⁵ cell/mL) and treated for 48 with one of the following treatments: 425 μM piperine, 80 μM TQ, combination of 425 μM piperine + 80 μM TQ and negative control which only contain MEM. Cells were processed according to the kit instructions. Briefly, cells were harvested, washed, and lysed using cell lysis buffer. Supernatants were collected and 100 μL was added to each well in the 96-well microplates coated with VEGF capture antibody, then incubated for 2.5 h. After washing, 100 μL of biotinylated detection antibody was added and incubated for 1 h followed by washing and adding 100 μL of horseradish peroxidase (HRP)-conjugated streptavidin with 45 min incubation. For color development 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution was added and incubated for 30 min in a dark place. Color intensity was measured at 450 nm after adding 50 μL of stopping solution.

All reagents were purchased from Sigma Chemical Company (St. Louis, MO, USA) unless otherwise stated. Isolated δ-tocotrienol (>95% purity) was generously provided by First Tech International Ltd. (Hong Kong). Antibodies for Akt (#9272), p-Akt (#9271, Ser473), PI3K (#4225), p-mTOR (#2971, Ser2448), mTOR (#2989), p-ERK 1/2 (#4337, Thr202/Tyr204), MEK (#8727), p-MEK 1/2 (#2338, Ser221/217), and α-tubulin (#2125) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies for HIF-1α (#Sc-8711), ERK1 (#Sc-93), and ERK2 (#Sc-154) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for p-p70S6K (#GTX530304, Ser424), p-eIF-4E (#GTX50268, Ser209), and p-4E-BP1 (#GTX61987, Thr37) were purchased from Gene Tex Inc. (Irvine, CA, USA). Goat anti-rabbit (#NEFB812001EA) and anti-mouse (#NEF822001EA) secondary antibodies were purchased from PerkinElmer Biosciences (Boston, MA, USA). Mouse VEGF ELISA kit was purchased from Sigma Aldrich (St. Louis, MO, USA).