Quantification of the interaction of neuronal adhesion molecules by flow cytometry using microbeads

Attila Tarnok; Ursel Noehrenberg; Stephan Schuhmacher; Hans-Juergen Volkmer

doi:10.1117/12.307073

1 May 1998 Quantification of the interaction of neuronal adhesion molecules by flow cytometry using microbeads

Attila Tarnok, Ursel Noehrenberg, Stephan Schuhmacher, Hans-Juergen Volkmer

Proceedings Volume 3256, Advances in Optical Biophysics; (1998) https://doi.org/10.1117/12.307073
Event: BiOS '98 International Biomedical Optics Symposium, 1998, San Jose, CA, United States

Abstract

Neuronal adhesion molecules expressed during embryogenesis are essential for axonal pathfinding and development of neural connections and can interact by homophilic or heterophilic adhesion. We used and further developed a simple flow cytometric (FCM) microbead method to assess these interactions. This method allows the detection of homo- or heterophilic interactions and gives estimates of the binding affinity. It is possible to determine binding domains of a molecule using site specific antibodies or protein fragments. Adhesion molecules of the immunoglobulin-superfamily or the tenascin (TN)-family isolated from chick brain were analyzed. Purified molecules were covalently or non-covalently coupled to microbeads. For investigation of heterophilic interactions different molecules were coupled to beads of different colors. Measurements were done on single laser FCM with UV or 488 nm excitation. From the measured particle numbers the numbers of beads in homo- and/or heterophilic aggregates were determined. From these values the percentage of beads in aggregates and the aggregate size was calculated. Homophilic interaction was found for the molecules NCAM, NgCAM and NrCAM. Heterophilic interaction were detected e.g. for: NrCAM/F11, TN-R/F11 and CALEB/TN-C and CALEB/TN-R. Using fragments of TN-R we found, that the third of 8 fibronectin type III domains bound to F11. The FCM results were confirmed by neurite outgrowth assays. Our data demonstrate that FCM analysis of microbead aggregation is an easy and reliable method to characterize protein interactions.

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Attila Tarnok, Ursel Noehrenberg, Stephan Schuhmacher, and Hans-Juergen Volkmer "Quantification of the interaction of neuronal adhesion molecules by flow cytometry using microbeads", Proc. SPIE 3256, Advances in Optical Biophysics, (1 May 1998); https://doi.org/10.1117/12.307073

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