RTCA background

RTCA background

Raw Data

Cells are placed in wells of plates with electrodes on the bottom which is usually kept in a cell culture incubator. The RTCA instrument measures impedance (related to electrical resistance), using an average voltage of 22 mV and µA  alternating currents (10 to 50 kHz) in wells with gold electrodes on the bottom. When cells cover the electrodes,  impedance increases. Impedance is affected by:

  • number of cells
  • extent to which cells adhere to plate
  • cell morphology

Short term changes are dues to morphology or adhesion changes, and commonly involve changes in calcium flux.

To help determine what is happening, you can visually examine cells, either in a parallel incubation, or using the E-plate VIEW 16.

The raw impedance is converted to the dimensionless Cell Index parameter to correct for well variations. Reading one plate takes about 4 seconds.

Plates

Acea's description of the plates is here. The wells, made of plastic, are 5 mm diameter and hold 270 µL although no more than 200 µL should be loaded. About 80% of the bottom is covered by gold electrodes layered on glass. Wells in the middle of the plate have higher impedance because the electrodes are larger in size because of their distance from the contact pins on the ends of the plates.

The plate for cell migration (CIM plate) has PET membranes, 5 mm diameter, with 8 µm pores at the bottom of 180 µL wells. Electrodes on the lower side of the membrane measure the presence of cells and thus migration of cells through the membrane. You can put Matrigel or other materials in the well.

The E-plate inserts have pores of 0.4 µm in the bottom of the wells.

The E-plate VIEW 16 is meant for visual examination of cells.  4 rows of microelectrode sensors are removed from the center of each well, reducing electrode coverage to 70% of the well, which reduces signal some, but normally signals are still adequate.

Running an experiment

If necessary, clean contact pads on plate with lint free wipes, or cloth moistened with 80% ethanol, or blow with compressed air. Problem with electrical contact will show on resistor plate assay, and may cause error messages.

When loading, use no more than 200 µL of cells, and avoid scratching the gold electrodes with pipet tips.

All wells should have cells or buffer in them.

Movement of plates can alter impedance, so after addition of reagents or other disturbance, the cells often need time to return to their previous state.