Our team has developed other models including
- Vascular permeability assay
- Human dermal papilla cell based inflammatory model – Alopecia
- Glucose Metabolism and oxidative stress
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
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
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.
Glucose Metabolism and oxidative stress
C2C12 are an immortalised mouse myoblast cell line. They a well-known model for metabolic disorders and are used to look at mitochondrial metabolism as well as glucose metabolism. They are a well documented at looking into the effects of ROS related oxidative stress.
Reactive oxygen species (ROS) are essential in a number of biological processes including metabolism, differentiation and proliferation. Oxidative stress is caused by the elevation of intracellular levels of ROS, which can cause damage to DNA, proteins, and lipids. Oxidative damage from ROS has been noted in several disease areas including cancer, aging, and metabolic conditions. It is important therefore to understand the role of ROS in a multitude of disease areas. ROS can be quantified in vitro, in live cells, by using the fluorogenic dye 2’,7’ –dichlorofluorescin diacetate (DCFDA) which diffuses in the cells and is converted to 2’, 7’ –dichlorofluorescein and measured by fluorescence spectroscopy .
Glucose Oxidase which is known to increase ROS through catalysing glucose to hydrogen peroxide was used as a control along with known ROS inducers tert-Butyl hydroperoxide (TBHP) and Palmitic acid (PA). PP2, a known inhibitor of Palmitic acid, was used to decrease the effect of PA on the release of ROS.
ROS Assay: GOx dose response
Prior to treatment C2C12 were differentiated to form mature myotubles and then incubated with DCFDA. C2C12 cells were treated with GOx at 7 concentrations and incubated 37°C in a humidified incubator. The fluorescence was measured by plate reader at 1 and 4 hours post treatment
ROS Assay: TBHP response
Prior to treatment C2C12 were differentiated to form mature myotubles and then incubated with DCFDA. C2C12 cells were treated with TBHP at 7 concentrations and incubated 37°C in a humidified incubator. The fluorescence was measured by plate reader at 1 and 4 hours post treatment
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