3.4. Real-Time Cell This method utilizes instrumentation (RTCA-DP, Roche Applied Electric Impedance Science/Acea Biosciences) which tracks electrical impedance sig-
Sensing nals, monitoring in real time the status of cells grown on microelectrode coated plates. The impedance readout is expressed in arbitrary units as “Cell Index,” which reflects changes in barrier function and permeability (8).

1. For each treatment/condition, plate enough wells to have at least triplicate conditions.
2. Add 100 ml EGM-2 to each well of the E-plate 16, insert plate into RTCA-DP instrument, and take background reading of selected wells using the RTCA software.
3. Seed appropriate number of endothelial cells into each well of the E-plate 16 (determined empirically, see Notes 1 and 9). In the case of HUVEC, 40,000 cells per well is sufficient to attain a completely confluent monolayer the next day.
4. Optional: Seed an equivalent density of cells (by area) on a coverslip for visual verification of monolayer (especially important if comparing different cell types).
5. Monitor impedance overnight, setting the software to take readings every 15–30 min.
6. On day 2, the impedance values will have reached a plateau, indicating a fully formed barrier.
7. At this point, one can add various compounds to appropriate wells and monitor changes in impedance over very short intervals, depending on the settings selected with the software (i.e., measurements every 1–15 min, for the desired total time interval).
8. Data can be represented graphically as Cell Index (CI) or Normalized Cell Index, which is useful when comparing effects upon the addition of drugs or compounds. To do this, select the timepoint immediately preceding the addition of compound as the reference value, which is set to “1.”
   Two examples are shown to validate this method as a way of detecting rapid changes in endothelial barrier function. Figure 3a shows increased barrier function in a dose-dependent manner upon the addition of a compound (8-CPT-2?-O-Me-cAMP)

Fig. 3. Real-time impedance analysis of endothelial barrier function. (a) Impedance was measured in triplicate wells every 15 min following the addition of various doses of the Epac-activating cAMP analog 8-CPT-2?-O-Me-cAMP, which is known to enhance barrier function. There is a dose-dependent increase in cell index, which is significant for all doses at t = 0.5 h and longer (p < 0.05). (b) After reaching steadystate index (t = 0), wells were treated with indicated junction-disrupting agents: EGTA (4 mM), clone75 Ab (VE-cadherin function-blocking AB), or thrombin (1 U/ml). Treatment with all compounds results in a rapid drop in cell index, indicating decreased barrier properties. Graph represents mean of triplicate wells ±SD. With the exception of the single data points thrombin and cl75 at t = 0.25 h, all remaining data points are significantly different from control (p < 0.05).
known to activate the Rap1 GTPase GEF, Epac (9), and positively affect the endothelial cell junction barrier (10, 11). Conversely, Fig. 3b illustrates the effects of various junction-disrupting treatments on endothelial barrier function. Treatments that can be used to disrupt endothelial cell junctions include calcium chelation by EGTA (12), thrombin treatment (5), and incubation with a VE-cadherin function-blocking antibody (13).