Quantification of gap junctional intercellular communication based on digital image analysis

• Published in: American journal of physiology. Regulatory, integrative and comparative physiology integrative and comparative physiology, 2009-08, Vol.297 (2), p.R243-R247
• Main Authors: Hofgaard, Johannes P, Mollerup, Sarah, Holstein-Rathlou, Niels-Henrik, Nielsen, Morten Schak
• Format: Article
• Language: English
• Subjects: Animals; Carbenoxolone - pharmacology; Cell Communication - drug effects; Cell Communication - physiology; Cell Line, Tumor; Cells; Connexin 43 - genetics; Connexins - pharmacology; Dextrans - metabolism; Digital imaging; Electroporation - methods; Fluorescence; Fluorescent Dyes - metabolism; Gap Junctions - drug effects; Gap Junctions - metabolism; Gene expression; Image Processing, Computer-Assisted - methods; Internet; Isoquinolines - metabolism; Microscopy, Fluorescence - methods; Neuroglia - metabolism; Octanols - pharmacology; Physiology; Rats; Software; Transfection
• ISSN: 0363-6119; 1522-1490
• DOI: 10.1152/ajpregu.00089.2009

The Danish National Research Foundation Centre for Cardiac Arrhythmia and Department of Biomedical Sciences, Faculty of Health Sciences, University of Copenhagen, Copenhagen N, Denmark Submitted 10 February 2009 ; accepted in final form 10 June 2009 Intercellular communication via gap junction channels can be quantified by several methods based on diffusion of fluorescent dyes or metabolites. Given the variation in intercellular coupling of cells, even under untreated control conditions, it is of essence to quantify the coupling between numerous cells to obtain reliable estimates of metabolic coupling. Quantification is often based on manual counting of fluorescent cells, which is time consuming and may include some degree of subjectivity. In this report, we introduce a technique based on digital image analysis, and the software for the analysis is presented together with a detailed protocol in the online supplemental material ( http://bmi.ku.dk/matlab_program/ ). Fluorescent dye was introduced in connexin 43-expressing C6 glioma cells by in situ electroporation, and fluorescence intensity was measured in the electroporated cells and in cells receiving dye by intercellular diffusion. The analysis performed is semiautomatic, and comparison with traditional cell counting shows that this method reliably determines the effect of uncoupling by several interventions. This new method of analysis yields a rapid and objective quantification process with a high degree of reproducibility. connexin; gap junction; electroporation; dye transfer Address for reprint requests and other correspondence: M. Schak Nielsen, The Danish National Research Foundation Centre for Cardiac Arrhythmias and Dept. of Biomedical Sciences, Blegdamsvej 3C, DK-2200 Copenhagen N, Denmark (e-mail: schak{at}mfi.ku.dk )