title: Antibody-Conjugated Single Quantum Dot Tracking of Membrane Neurotransmitter Transporters in Primary Neuronal Cultures.
abstract: Single particle tracking (SPT) experiments have provided the scientific community with invaluable single-molecule information about the dynamic regulation of individual receptors, transporters, kinases, lipids, and molecular motors. SPT is an alternative to ensemble averaging approaches, where heterogeneous modes of motion might be lost. Quantum dots (QDs) are excellent probes for SPT experiments due to their photostability, high brightness, and size-dependent, narrow emission spectra. In a typical QD-based SPT experiment, QDs are bound to the target of interest and imaged for seconds to minutes via fluorescence video microscopy. Single QD spots in individual frames are then linked to form trajectories that are analyzed to determine their mean square displacement, diffusion coefficient, confinement index, and instantaneous velocity. This chapter describes a generalizable protocol for the single particle tracking of membrane neurotransmitter transporters on cell membranes with either unmodified extracellular antibody probes and secondary antibody-conjugated quantum dots or biotinylated extracellular antibody probes and streptavidin-conjugated quantum dots in primary neuronal cultures. The neuronal cell culture, the biotinylation protocol and the quantum dot labeling procedures, as well as basic data analysis are discussed.
authors.affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN, 37235, USA.|Interdisciplinary Materials Science Program, Vanderbilt University, Nashville, TN, 37235, USA.|Department of Pharmacology, Vanderbilt University, Nashville, TN, 37235, USA.
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Bailey
authors.affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN, 37235, USA.
authors.forename: Oleg
authors.initials: O
authors.lastname: Kovtun
authors.affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN, 37235, USA. sandra.j.rosenthal@vanderbilt.edu.|Department of Pharmacology, Vanderbilt University, Nashville, TN, 37235, USA. sandra.j.rosenthal@vanderbilt.edu.|Vanderbilt Institute for Nanoscale Science and Engineering, Vanderbilt University, Nashville, TN, 37235, USA. sandra.j.rosenthal@vanderbilt.edu.|Department of Physics and Astronomy, Vanderbilt University, Nashville, TN, 37235, USA. sandra.j.rosenthal@vanderbilt.edu.|Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, 37235, USA. sandra.j.rosenthal@vanderbilt.edu.|Materials Science and Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA. sandra.j.rosenthal@vanderbilt.edu.
authors.forename: Sandra J
authors.initials: SJ
authors.lastname: Rosenthal
mesh_terms: D000465:Algorithms; D000818:Animals; D005455:Fluorescent Antibody Technique; D018796:Immunoconjugates; D008962:Models, Theoretical; D057054:Molecular Imaging / Q000379:methods*; D009474:Neurons / Q000378:metabolism*; D050479:Neurotransmitter Transport Proteins / Q000378:metabolism*; D061251:Primary Cell Culture; D045663:Quantum Dots; D051381:Rats; D000072760:Single Molecule Imaging / Q000379:methods; D013223:Statistics as Topic / Q000379:methods
chemical_list: D018796:Immunoconjugates; D050479:Neurotransmitter Transport Proteins
keywords: Antibody; Neurotransmitter transporter; Quantum dot; Single particle tracking