Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

The overall goal of the following experiment is to quantify rapid changes in cell matrix adhesion in endothelial cells exposed to matrix modifying agents in real time using cell substrate impedance and live cell imaging. In this experiment, the heme enzyme myelo peroxidase is employed as a pathophysiologically relevant matrix modifying agent. This is achieved by first coating surfaces with fibronectin to provide an adhesive matrix substrate.

Myo peroxidase or MPO is then allowed to bind to the fibronectin. Next suspended endothelial cells are seated onto the surfaces and incubated to attain maximal cell attachment and spreading onto the fibronectin. Then the cells are exposed to hydrogen peroxide in order to initiate NPO mediated fibronectin oxidation.

The subsequent rapid changes in cell matrix adhesion are then quantified in real time using cell substrate impedance and live cell imaging analysis. The results show that rapid losses in endothelial cell matrix adhesion triggered by NPO mediated fibronectin oxidation can be quantified in real time with high temporal resolution and in a label free manner by cell substrate impedance and live cell imaging. Well, the advantage of these techniques is they can quantify changes in cell matrix adhesion continuously with high temporal resolution, and this means that they can provide important new insights into how matrix modifications drive downstream changes in cell morphology and adhesion dependent signaling processes.

The protocols are of broader significance in that they can be readily adapted by substituting MPO for other matrix modifying agents, such as matrix to gating, proteases, or by using adherent cell types other than endothelial cells such as epithelial cells. To begin, add 80 microliters per well of fibronectin to coat 96 well gold micro electrode arrays for cell substrate impedance measurements, or add two milliliters of fibronectin to glass bottom cell culture dishes for live cell imaging analysis. Incubate the arrays and or the culture dishes at 37 degrees Celsius for two hours.

Then to block the surfaces, add 0.2%BSA and incubate at 37 degrees Celsius for an additional two hours. Next, add purified human my peroxidase in HBSS and incubate for 30 minutes. Then use HBSS to wash the surfaces twice to remove any unbound MPO.

Next, see 250, 000 cells per milliliter of suspended endothelial cells prepared according to the text protocol Onto 96 well cells substrate impedance micro electrode arrays with a native or MPO bearing fibronectin coated surfaces. For the 35 millimeter glass bottomed cell culture dishes, add two milliliters of 500, 000 cells per milliliter. Immediately after seeding the cells mount the micro electrode array plates onto the cell substrate impedance incubator port at 37 degrees Celsius and 5%carbon dioxide.

And take a blank readings to initiate continuous acquisition of cell index data. Transfer the 35 millimeter glass bottom cell culture dishes to a 35 degrees Celsius 5%carbon dioxide incubator. Incubate for two hours to allow maximal cell attachment and spreading.

After the incubation, pause the cell index readings and briefly remove the microelectrode array plate or remove the glass bottom dish from the incubator, aspirate the snat and add warmed HBSS to examine the effects of inhibitors, modulators of MPO, catalyzed reactions or cell signaling inhibitors. Add them to the cultures. At this point, immediately place the array plate back into the incubator port or the glass bottom dish into the incubator and allow the cells to equilibrate for 30 minutes.

To initiate NPO catalyzed hypochlorous acid dependent fibronectin oxidation and de adhesion for cell impedance studies, remove the microelectrode array plate from the incubator and pause the cell index measurements. Add hydrogen peroxide to the HBSS and gently mix by repeated pipetting immediately remount to the microelectrode array back into the incubator port and restart cell index readings for live cell imaging studies using 35 millimeter glass bottomed dishes. After removing the dish from the incubator, mounted onto a 37 degree Celsius heated stage of an inverted confocal microscope, equipped with a 63 x water objective lens.

And DIC optics For recording live cell movies, focus on the cells and optimize DIC optics according to the guidelines referenced here. Initiate DIC movie and record baseline readings of untreated cells for one minute, then add hydrogen peroxide and gently mix by repeated pipetting, refocus the microscope and continue recording DIC movies of the treated cells for the required period of time. To analyze cell substrate impedance data, begin by exporting the data into a spreadsheet for cell d adhesion.

Studies normalize data by setting values recorded immediately prior to the initiation of NPO mediated fibronectin oxidation to one present data as plots of normalized cell index versus time to analyze live cell imaging data. Open DIC live cell imaging movies recorded immediately prior to and after the initiation of MPO mediated fibronectin oxidation in an image analysis program such as Image J in at least two separate DIC movies, randomly select multiple cells and measure their projected area in sequential frames by manually tracing their membrane edge and quantifying the number of enclosed pixels. Finally present the data as a plot of normalized cell area versus time the seating of endothelial cell suspensions onto fibronectin or MPO bearing fibronectin results in maximal cell attachment and spreading within two hours as judged by a plateauing of cell index values in the cell substrate.

Impedance measurements, once maximal cell adhesion is established on native or NPO bearing fibronectin, targeted fibronectin oxidation mediated by NPO catalyzed hypochlorous acid and initiated by the addition of NPOs cos substrate Hydrogen peroxide causes a rapid decrease in the surface area of cell matrix contact measured by cell substrate impedance and by live cell imaging as shown here, the rapid cellular de adhesion apparent in the endothelial cells bound to NPO bearing fibronectin in response to hydrogen peroxide treatment is absent in cells treated with hydrogen peroxide alone. Inhibition of myosin two motor function with BLEs statin inhibits the rate of endothelial cell de adhesion measured by cell substrate impedance, and by live cell imaging identifying that cellular de adhesion and contraction in response to MPO catalyze subcellular matrix oxidation is driven by ACTO myosin tensile forces. As these methods can accurately measure very rapid changes in cell adhesion, they can give critical insight into the causal relationships between adhesion changes and downstream changes in adhesion dependent signaling processes such as SARK phosphorylation or focal adhesion signaling, which can be readily measured in parallel experiments over time using Western blotting.