Limitations of the scrape-loading/dye transfer technique to quantify inhibition of gap junctional intercellular communication

Abstract

Gap junctional intercellular communication (GJIC) is recognized as playing an important role in normal cell proliferation and development. Chemically induced alteration of GJIC has been proposed to be associated with abnormal cellular growth and/or tumor promotion. Several in vitro assays are currently used to determine the effects of chemicals on GJIC between cultured mammalian cells. One of these assays, the scrape-loading dye transfer (SL/DT) technique, is based on monitoring the transfer of the fluorescent dye Lucifer yellow from one cell into adjacent cells via functional gap junctions. The objective of our study was to evaluate and compare various approaches for quantifying results obtained with the SL/DT technique. Confluent cultures of either WB rat liver epithelial cells or LC-540 rat leydig cells were exposed to the animal tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), solvent (0.1% ethanol), or culture medium for one hour at 37 degrees C prior to analysis of GJIC. Inhibition of dye transfer was clearly evident following TPA exposure. Quantification of this dye transfer was assessed via four approaches: manually counting the number of labeled cells; measuring the distance of dye travel from the scrape line; quantifying the amount of cellular dye uptake; and determining the distribution of dye away from the scrape line. Our results suggest that while the SL/DT technique can be effectively used as a tool to determine the qualitative presence or absence of GJIC, its use in quantifying changes in GJIC following chemical exposure is limited. Since concentration-dependent responses are critical in chemical testing, application of the SL/DT method should be restricted to a screening assay for qualitatively assessing the presence or absence of GJIC.

Changes in cell morphology affect the quantification of intercellular communication

“…Mckarns and Doolittle (1992) compared different methods and proposed the use of SL/DT only to determine the qualitative presence or absence of GJIC. In contrast, Opsahl and Rivedal (2000) had compared the use of both SL/DT and microinjection as tools for GJIC quantification and they had demonstrated that scrape loading combined with image analysis can even offer advantages on the microinjection.…”

Rat endometrial stromal cells express the gap junction genes connexins 26 and 43 and form functional gap junctions during in vitro decidualization

“…7). Because scrape-loading is a qualitative method, quantitative comparisons were not made [37]. The dye-coupling experiments suggest that endometrial stromal cells develop functional gap junctions early during culture (3 h) and that they remain present throughout the culture period (up to 72 h).…”

Development of a reference dose for the persistent congeners of weathered toxaphene based on in vivo and in vitro effects related to tumor promotion

“…Disruption of gap junctional communication can be measured by intercellular transfer of the dye Lucifer yellow following application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) (McKarns and Doolittle, 1982). A study by Kang et al (1996) showed that noncytotoxic concentrations of TT inhibited GJIC in normal human breast epithelial cells in a dose-dependent way after 90 min of exposure.…”

“…This dye has been used for a number of years to observe disruption of gap junctional communication and electrical junctions between nerve cells. (e.g., McKarns and Doolittle, 1982;Spencer and Satterlie, 1980). 2,3,7,8-TCDD was used as a positive control.…”