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Assays

Assays

Barrier integrity assay

The principle of barrier integrity assays in OrganoPlates®

In vivo, endothelial and epithelial monolayers form barriers. These barriers play key roles in regulating the movement of molecules and cells within tissues. Certain disease states, such as inflammation, can compromise the integrity of these barriers. Consequently, it is important that we can measure the barrier integrity in vitro.

The OrganoPlate® allows you to study the barrier integrity of endothelium and epithelium in real-time and with unsurpassed sensitivity. To this end, tissues are seeded against the ECM gel in the OrganoPlate® to form tubular structures with tight junctions. Within a few days, these perfused tubules become fully polarized and leak-tight.

You can assess the barrier integrity by perfusing the tubule with a fluorescent dye. Using a standard fluorescent microscope, you can monitor the movement of the fluorescent dye through the barrier tissue. The barrier integrity is quantified by comparing the fluorescent intensity in the ECM channels to the intensity in the perfusion channels with endothelial or epithelial tubules.

Immunohistochemistry with fluorescent detection

Co-culture of astrocytes and HUVECs in the OrganoPlate®

Immunofluorescence is a technique that is uses fluorescently labeled antibodies or molecules to visualize specific cellular biomolecules expressed in cells. A primary antibody binds to the target molecule. A secondary antibody, conjugated with a fluorophore, binds to the primary antibody. Fluorescent imaging allows you to visualize and quantify the distribution of target molecules in the OrganoPlate®.

The OrganoPlate® allows immunofluorescent detection of biomolecules inside the plate. The 96 tissue chips support easy comparison of biomolecules expressed in a multitude of differentially exposed tissues. The light intensity of the tagged antibodies is measured by confocal fluorescence microscopy. You can quantify the fluorescence intensity using image analysis software.

Figure: Co-culture with astrocytes and HUVECs stained by Immunohistochemistry in the OrganoPlate®.

Neuronal activity using calcium imaging

A fluorescent calcium indicator (Fluo-4) is used to study the electrophysiological activity of single neurons and the network as a whole. Fluo-4 is a cell permeant dye that emits a fluorescent signal upon binding to calcium. The calcium indicator is taken up by the cells and a strong increase in fluorescent signal is observed when intracellular calcium levels rise, an event associated with neuronal firing. Imaging these fluctuations in fluorescent signal over time allows us to evaluate the activity of the cells and the network and investigate the effect of various compounds on electrophysiology. 

The OrganoPlate® supports real-time imaging of neuronal firing inside the plate. First, you seed mature iPSC-derived neurons (with or without supporting cell types) in the OrganoPlate®. In just a couple of days, these neurons form active neuronal networks. Using a Fluo-4 cell permeant dye you can measure the dynamic calcium flux within neurons and neuronal tissue in the OrganoPlate®.

Figure: Calcium image recording of a neuronal culture after 6 days in the OrganoPlate®. 

Cell viability assays

Live dead staining in the OrganoPlate®.

Determining the viability of cultured cells is crucial for establishing 3D tissue culture models. Cell viability can be assessed using various indicators, such as plasma membrane integrity and metabolic activity. By using cell viability assays in the OrganoPlate®, you can also test the cytotoxicity of compounds in 3D tissue models. The following cell viability assays have been validated in the OrganoPlate®.

  • The RealTime-Glo™ MT Cell Viability Assay (Promega) is a method to measure cell viability in real-time. The non-lytic nature of this assay allows the cells to be used for further applications. The number of viable cells in culture is determined by measuring the reducing potential of cells using a substrate that is linked to a luminescent signal.
  • The Alamar Blue assay (resazurin, Sigma Aldrich) is a non-lytic assay, also based on the reducing potential of living cells. The cell permeable, non-fluorescent compound resazurin is added to the culture medium in the OrganoPlate®. Viable cells convert resazurin into red fluorescent resorufin, which enables quantitative measurement by fluorescence microscopy.
  • The fluorescent Live/Dead assay (Life Technologies) is an endpoint measurement for cells cultured in the ECM. The cells are stained with three different fluorescent labels (NucBlue Live-, Calcein-AM- and NucRead Dead stains), to distinguish living from dead cells. Live/dead measurements are visualized by fluorescence microscopy.
Figure: Live dead staining in the OrganoPlate®. 

Other functional assays

  • Apoptosis induction (IncuCyte™ Caspase-3/7) (in-plate assay)
  • Metabolic activity: (CYP1A activity) (fluorescent in-plate assay)
  • Functional transport (Pgp, SGLT2, MRP2 transport) (fluorescent in-plate assays)
  • Gene expression analysis (quantitative RT-PCR) (off-plate assay)
  • Fluorescence recovery after photobleaching (FRAP) assay (in-plate assay)
  • Secretion/ efflux assay:
    - Human Albumin ELISA assay (Bethyl) (off-plate assays)
    - Urea colorimetric assay (BioVision) (off-plate assays)
  • Proliferation assay (EdU assay) (in-plate assays)
  • Monocyte attachment and transmigration in blood vessel (fluorescent in-plate assays)
  • Induction and/or inhibition of angiogenesis (in-plate assays)
  • Protein expression levels (Western blot) (off-plate assay)
  • Differentiation status (Alkaline phosphatase activity) (in-plate assay)
  • Mitochondrial potential (TMRM assay, JC-1 assay) (in plate assay)