Our team has developed other models including Vascular permeability assay and a human follicle dermal papilla cell based inflammatory model.
HUVECs were allowed to form a monolayer, prior to treatment with the VEGFR inhibitor Axitinib (AX) and VEGF-A. Untreated control was also included as part of the analysis. (A) FITC-Dextran mediated measure of permeability. VEGF-A treatment resulted in an increase in monolayer permeability as indicated by the increase in fluorescence output (RFU) relative to the untreated condition. Pre-treatment with AX reduced the fluorescence output despite the addition of VEGF-A, showing that AX reverses the permeabilisation effect of VEGF-A. (B) Calcein-AM staining of HUVEC cells after drug treatment. A tight HUVEC monolayer was formed in the untreated condition but upon treatment with VEGF-A, the cellular arrangement has changed indicating movement and motility. Pre-treatment with AX maintained the integrity of the monolayer cell arrangement as that found in the untreated condition.
Human Dermal Papilla Cells and Inflammation
Human Follicle Dermal Papilla Cells were treated with Minoxidil and Emodin for 48 hours. Cell lysates were then analysed for TGFβ1, TGFβ2, IGF1 and VEGFA gene expression using Quantigene™ Multiplex Assay. Changes in expression levels for each treatment condition were normalised to the house keeping gene and compared to the vehicle control (***p<0.001, **p<0.01, *<p0.05; n=3±SEM).
HDMEC membrane permeability on transwell model
Permeability of primary Human Dermal Microvascular Endothelial Cells (HDMEC) on a transwell model. HMDEC were seeded onto gelatin-coated HTS96 well-plate format transwells for 4 days, followed by 24h treatments. TEER was measured before and after treatments, and FITC-dextran permeability was determined after treatments. A,B) Treatment with TNFα caused a reduction in TEER and increase in FITC-permeability, thereby increasing HDMEC permeability. This was partially reversed by co-treatment with anti-TNFα antibody Adalimumab C,D) Treatment with VEGFA resulted in a decrease in HDMEC permeability, as seen by an increase in TEER, which was accompanied by reduction in FITC-dextran permeability.
Data expressed as mean +/- SD (N=3), statistical comparison was performed by one-way ANOVA with Dunnett’s test for multiple comparison against Vehicle controls, and unpaired t-test between TNFα +/- ADA (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).
Caco2 calcium permeability on transwell model
Assay to quantify Caco2 permeability to calcium in a transwell model. A) A colorimetric assay kit was used to determine the amount of CaCl2 in HBSS buffer (without Ca2+ or Mg2+), showing a dose-dependent signal increase between 0-15 μg. B) Caco2 cells were seeded in 0.4μm pore-sized transwells for 4 days until a TEER value of 400 Ω.cm2 was reached, followed by addition of 60 μg of CaCl2 in HBSS buffer to the apical compartment. Quantification of CaCl2 in apical and basal chambers was performed at 4 time points, with empty transwells and HBSS only being used as controls. C) While most of the CaCl2 added in empty transwells diffuses through to the basal compartment, there is a gradual permeation of CaCl2 through Caco2-seeded transwells over time, as shown by increased % of calcium in the basal compartment, reaching approximately 10% (6μg) after 180 minutes.
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