HUVECs were cultured in Transwell chambers (0.4 μm pore polycarbonate filters; Costar, Cambridge, Miss.). After reaching confluence, the medium was replaced with the previously-collected conditioned medium (0.3 ml in the upper chamber and 1 ml in the lower chamber). Horseradish peroxidase molecules (type VI-A, 44 kDa; Sigma-Aldrich) at a concentration of 0.126 μM were then added to the upper compartment. After incubating for 1 hr, the medium in the lower compartment was assayed for enzymatic activity using a photometric guaiacol substrate assay (Sigma-Aldrich).
Photometric guaiacol substrate assay
Manufactured by Merck Group
Sourced in United States
The Photometric guaiacol substrate assay is a laboratory equipment product used to measure the activity of certain enzymes. It provides a quantitative analysis of the enzyme's catalytic function by monitoring the change in absorbance of a colored product formed during the enzymatic reaction.
Automatically generated - may contain errors
4 protocols using photometric guaiacol substrate assay
1
Permeability Assay for HUVECs
2
HRP Permeability Assay in HUVECs
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HUVECs were cultured in Transwell chambers (0.4 μm pore polycarbonate filters; Costar, Cambridge, MA, USA). After reaching confluence, the medium was replaced with the CM (0.3 mL in the upper chamber and 1 mL in the lower chamber). Horseradish peroxidase molecule (Sigma-Aldrich, Saint Louis, MO, USA) was added to the upper compartment at a concentration of 0.2 µM. After incubation for 1 h, the medium in the lower compartment was assayed for enzymatic activity while using a photometric guaiacol substrate assay (Sigma-Aldrich).
3
Permeability Assay of Endothelial Barrier
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HUVECs were cultured in Transwell chambers (0.4 ml pore polycarbonate filters; Costar, Cambridge, Miss., USA). After reaching cell confluency, the medium was replaced with CM (0.3 ml in the upper chamber and 1 ml in the lower chamber). Horseradish peroxidase molecules (type VI-A, 44 kDa; Sigma-Aldrich) at a concentration of 0.126 µM was added to the upper compartment. After incubation for 1 h, the medium in the lower compartment was assayed for enzymatic activity using a photometric guaiacol substrate assay (Sigma-Aldrich).
4
HUVEC Monolayer Permeability Assay
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The HUVECs were seeded on Transwell chambers with 0.4 μm pore-sized polycarbonate membranes (Costar, Cambridge, MA, USA) and grown to confluency to form a monolayer. The HUVECs were pretreated with medium alone (control), VEGF (50 ng/ml) alone, VEGF (50 ng/ml) plus low-dose IL-10 (10 pg/ml), VEGF (50 ng/ml) plus high-dose IL-10 (100 pg/ml), or high-dose IL-10 (100 pg/ml) alone. Horseradish peroxidase (HRP; Sigma-Aldrich) at a concentration of 0.1 μM was added to the upper chamber. HRP diffusion through the HUVEC monolayer was measured and used to represent permeability. After incubation for 1 h, the medium in the lower chamber was assayed for HRP activity using a photometric guaiacol substrate assay (Sigma-Aldrich). The permeability was calculated by dividing the HRP concentration in the lower chamber medium by the initial HRP concentration in the upper chamber. The results from all the wells were normalized by the control wells and shown as percentages.
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