VO-CRO In Vitro Readouts
Using our in vitro models, we can assess any number of different readouts. These can include assays to determine RNA or protein levels in cells, evaluation of activation of cell signaling pathways or more complicated behavior assays to study cell adhesion, permeability, proliferation, migration, apoptosis, and other behaviours.
Our scientists are experts in molecular biology techniques, particularly related to quantification of mRNA and protein levels. We routinely perform quantitative RT-PCR, immunoblotting, ELISA, and RNA sequencing. We also have well-established methods for assessing target induction under disease-relevant conditions. All techniques are applicable to both cell- and tissue-derived samples.
We are capable of performing common molecular biology techniques or partnering with the Vanderbilt VANTAGE core to sequence RNA. These assays can be used with in vitro cell assays or with tissue derived from animal-based experiments.
RNAseq: RNA-sequencing can be used to develop an overall impression of differential mRNA gene expression. This is a valuable tool for determining an unknown mechanism of action.
qRT-PCR: Quantitative reverse transcription PCR (qRT-PCR) is used to analyze gene expression. This allows us to assess the drug mechanism of action and drug activity.
Multiplex and mass spectroscopy methods are used to define the proteomic profile of ocular cells, fluids, or tissues, while ELISA and immunoblotting are used to determine the levels of specific proteins in those samples.
In vitro cell behavior can help elucidate drug mechanism of action, and can be a cost-effective method for testing drug efficacy. Our scientists have extensive experience with a wide variety of in vitro behavior assays.
Proliferation: Endothelial cells proliferate in response to angiogenic stimuli. We assay proliferation by incorporation of bromodeoxyuridine into DNA.
Migration: Endothelial cell migration can be measured by a variety of methods, including scratch or stencil assays or transwell assays designed to determine the influence of chemoattractants. Assay characteristics can be customized based on specific needs.
Tube formation: We assay tube formation by incubating endothelial cells on Matrigel™ coupled with computer-assisted image analysis. Time-lapse photography is available to visualize this process.
Cell adhesion: The interaction between endothelial cells and circulating polymorphonuclear cells is an essential marker of vascular inflammation. We measure this event using either a static adhesion assay or a parallel plate flow chamber assay in which cell velocity and stasis can both be quantified.
Permeability: The tight junctions between retinal endothelial cells are an essential part of the blood retina barrier. Dysfunction of this barrier can result in retinal edema. To measure EC monolayer permeability we use a trans-endothelial electrical resistance (TEER) assay. In addition, qualitative information on junctional complex function can be determined by ICC directed at junctional complex components.
Apoptosis/protection: Endothelial cell survival is vital to maintain the health of the vasculature. We assay cell survival by a wide variety of different apoptosis assays, including the incorporation of flow cytometry.