Monitoring viral-induced cell death using electric cell-substrate impedance sensing.

Using an electrical measurement known as electric cell-substrate impedance sensing (ECIS), we have recorded the dynamics of viral infections in cell culture. With this technique, cells are cultured on small gold electrodes where the measured impedance mirrors changes in attachment and morphology of cultured cells. As the cells attach and spread on the electrode, the measured impedance increases until the electrode is completely covered. Viral infection inducing cytopathic effect results in dramatic impedance changes, which are mainly due to cell death. In the current study, two different fish cell lines have been used: chinook salmonid embryonic (CHSE-214) cells infected with infectious pancreatic necrosis virus (IPNV) and epithelioma papulosum cyprini (EPC) carp cells infected with infectious hematopoeitic necrosis virus (IHNV). The impedance changes caused by cell response to virus are easily measured and converted to resistance and capacitance. An approximate linear correlation between log of viral titer and time of cell death was determined.

Melanogenesis and evidence for melanosome transport to the plasma membrane in a CD83 teleost leukocyte cell line.

Visceral organs of ectothermic vertebrates harbour melanin-containing leukocytes termed melanomacrophages. These cells are thought to participate in immune reactions and free-radical trapping. In teleosts, the melanin-producing ability of melanomacrophages has hitherto not been confirmed by molecular techniques. Here, a leukocyte marker and the apparatus for melanosome production and transport were investigated in an Atlantic salmon (Salmo salar) pronephros-derived mononuclear leukocyte (SHK-1) cell line. The SHK-1 cells expressed transcripts specific for a mammalian CD83 homologue, a standard surface marker for activated or differentiated dendritic cells, and dopachrome tautomerase/tyrosinase-related protein-2, a melanocyte specific enzyme essential for melanin production. Reduction potential of melanin or its precursors was demonstrated histochemically after prolonged cultivation. Ultrastructural investigations revealed tyrosinase and acid phosphate activity in identical organelles and BSA-gold co-localized with multilamellar melanosomes after 2 h internalization. Apparently, melanosomes were transported and released through periodically occurring tubules fusing with the plasma membrane. Video monitoring revealed filopodia and macropinocytosis. These results showed that the SHK-1 cell line is capable of melanogenesis and melanosome secretion. Melanin-producing cells in teleost pronephros may represent a distinct CD83(+) leukocyte population consisting of phylogenetically relict multifunctional cells. This is the first report of a melanin-producing leukocyte cell-line.

Combining optical and electrical impedance techniques for quantitative measurement of confluence in MDCK-I cell cultures.

A new method combining optical and electrical impedance measurements is described that enables submicroscopic cell movements to be monitored. The cells are grown on small gold electrodes that are transparent to light. This modified electrical cell-substrate impedance sensor (ECIS) allows simultaneous microscopic recording of both growth and motility, thus enabling cell confluence on the electrodes to be systematically correlated to the impedance in regular time intervals of seconds and for extended periods of time. Furthermore, the technique provides an independent measure of monolayer cell densities that we compare to calculated values from a theoretical model. We have followed the attachment and spreading behavior of epithelial Madin-Darby canine kidney strain I (MDCK-I) cell cultures on microelectrodes for up to 40 h. The studies reveal a high degree of correlation between the measured resistance at 4 kHz and the corresponding cell confluence in 4- to 6-h intervals with typical linear cross-correlation factors of r equaling approximately 0.9. In summary, the impedance measured with the ECIS technique provides a good quantitative measure of cell confluence.