Hca-F cells were incubated with fucoidan (0, 100, 200 and 400 µg/mL) and CoCl2 (100 µM) for 24 h, and the collected supernatants were detected by ELISA, as previously reported [24 (link)]. Ninety six-well plates were coated with polyclonal anti-VEGF-C and anti-HGF (1:1000) antibodies, respectively. The plates were read on a Multiskan Ascent photometer (Thermo Fisher Scientific, Carlsbad, CA, USA) at 450 nm.
Multiskan ascent photometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Multiskan Ascent photometer is a versatile laboratory instrument designed for absorbance-based measurements. It provides accurate and reliable data for a variety of applications, including enzyme-linked immunosorbent assay (ELISA), colorimetric assays, and kinetic studies. The Multiskan Ascent features multiple wavelength capabilities and supports a range of microplate formats, allowing researchers to adapt the instrument to their specific experimental needs.
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5 protocols using multiskan ascent photometer
1
Fucoidan Effects on VEGF-C and HGF
2
Quantifying TGFβ Secretion in miRNA-Transfected HUVECs
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Quantikine ELISAs from R&D Systems (Minneapolis, MN) were used for the determination of TGFβ1 (cat# SB100B) and TGFβ2 (cat# SB250) in cell culture supernates. HUVECs were transfected with 20 nM of either control miRNA mimic or miR-30b mimic and were maintained in MCDB 131 with 0.5% FBS for 24 hours prior to collection of supernates. Cell culture supernates were collected and debris was removed through centrifugation at 1,200 x g for 5 minutes followed by storage of the supernates at -80°C. ELISAs were run according to manufacturer’s instructions in duplicate and absorbance was read at 450 nm with correction at 570 nm on a Multiskan Ascent photometer (Thermo Scientific, Rockford, IL). A standard curve was created for the determination of TGFβ concentrations by interpolation. Basal levels of TGFβ in culture media alone was subtracted as background from each cell culture sample.
3
Measuring Notch Signaling Activity
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Transfected and heat-shocked HeLa cells were washed with PBS and lysed with passive lysis buffer (Promega), which had been diluted to 1× with mq H2O. For measuring luciferase activity from cells expressing the 12×CSL luciferase reporter gene, 2 µl of sample was mixed with 18 µl of passive lysis buffer, and the luciferase activity was measured from triplicates with a luminometer (Thermo Scientific). The 12×CSL luciferase reporter has been described earlier [48 (link), 49 (link)]. For measuring β-galactosidase activity, a mixture of 10 µl of sample and 240 µl of ONPG (ortho-nitrophenyl-β-galactoside) buffer was incubated for 30 min at 37 °C, and the absorbance was measured from triplicates with a Multiskan Ascent photometer (Thermo Scientific) at 420 nm. Notch activity was calculated by relating the luciferase expression levels to the expression levels of β-galactosidase. To verify equal expression levels of Notch, lysates for immunoblotting were taken prior to adding passive lysis buffer. The lysates were immunoblotted against Notch with α-Notch1 C20 (Santa Cruz Technology), and α-actin (Cell Signaling) was used as a loading control. Values shown in the figures are statistically significant at ***P < 0.001 or *P < 0.05 as indicated. Values indicate the average of at three independent experiments.
4
VEGF-C and Phospho-Flk-1/Flt-4 Quantification
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MDA‐MB‐231 cells were incubated with fucoxanthin (25, 50, 100 μmol/L) for 24. The supernatant collected was analysed by ELISA. Each 96‐well plate was coated with polyclonal anti‐VEGF‐C (1:1500). Staining was measured at 450 nm on a Multiskan Ascent photometer (Thermo Fisher Scientific). Relative amounts of phospho‐Flk‐1/Flt‐4 were determined with a phospho‐Flk‐1/Flt‐4 cell‐based colorimetric ELISA kit (ImmunoWay Biotech, Plano, TX).
5
Quantification of VEGF-C and HGF by ELISA
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VEGF-C and HGF levels were evaluated by ELISA in 96-well plates coated with polyclonal anti–VEGF-C (1∶1000), anti-HGF (1∶100) antibodies diluted in 50 mM carbonate buffer (pH 9.0), respectively. Blocking buffer (PBS containing 1% BSA and 0.02% azide) was added to block non-specific protein binding. Different concentrations of VEGF-C and HGF growth factors were used to draw standard curve, respectively. Culture medium samples collected from the western blot assay were diluted 1∶100 in blocking buffer were added. The plates were washed with PBS containing 0.05% Tween-20. Rabbit anti-mouse HRP-conjugated secondary antibody (1∶5000 in blocking buffer) was added, respectively. Tetramethylbenzidine HRP color development solution (KeyGen Biotech) was added for 10 min development in a darkroom. The reaction was stopped by adding 50 µl 2 M H2SO4. The plates were read on a Multiskan Ascent photometer (Thermo Fisher Scientific) at A450.
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