Indirect Visualization of Protein Loading in Living DRG Neurons by Concomitant Trituration Loading of Fluorescence-Labeled Dextran

抄録

We established a simple method to evaluate the ratio of protein loading in cultured dorsal root ganglion (DRG) neurons. Fluorescein isothiocyanate (FITC)-conjugated dextran was used as a tracer of protein loading. Antibodies and FITC-dextran were concomitantly introduced into chick DRG neurons using a trituration method (by which macromolecules can be mechanically introduced into cell populations using pipettes). A correlation between the antibody loading and the fluorescence of FITC-dextran was determined quantitatively. Complete retention of the loaded antibody for 3 hours after trituration was confirmed immunocytochemically in approximately 71% of the FITC-dextran-positive cells. The ratio of the loaded antibody correlated quantitatively with the fluorescence intensity of FITC-dextran in a single cell. Trituration loading of proteins can be indirectly visualized and quantitatively evaluated by the fluorescence of FITC-dextran concomitantly loaded with proteins into neurons in primary culture. We use this method to monitor protein loading in living cultured DRG neurons.