This protocol describes the characterization and application of a new pH-sensitive fluorescent lipid probe, specific for cell membranes, which allows researchers to monitor exo and endocytosis in live cells. Most previous reporters on membrane protein based on pH-sensitive green fluorescence protein, which is indirect. As a lipid mimetic, ND6 integrate into the cell membrane, and thus faithfully report lipid membrane trafficking.
ND6 probe becomes fluorescent only if embedded in biological membrane environment. The fluorescence intensity of that probe correlates with protonation and deprotonation of the piperazine head group. Thus, it fluoresces much stronger at lower pH.
Exocytosis and the endocytosis are ubiquitous processes underlying the secretion and uptake of many signaling molecules. Therefore, ND6 can be used to study some fundamental questions in cell biology and the biomedical sciences. Demonstrating the procedure will be Haley, Oliver, and Jonathan, students from my lab.
Begin by preparing an imaging chamber that allows temperature control and solution input output for live cell fluorescence imaging. Set up a programmable device to switch the perfusion solutions and deliver the electric stimulus at defined time points during imaging. Weigh out an appropriate amount of ND6.
Dissolve it in dimethyl sulphoxide, and allow it to solubilize at room temperature. Then sonicate the solution briefly. Filter the crude stock solution using a 0.22 micrometer filter to remove large dye aggregates.
Using a conventional or micro volume spectrophotometer, determine the dye concentration at 405 nanometers. Before application, dilute the stock solution to a concentration of one milligram per milliliter using the bath solution. Keep the stock solution at room temperature in the dark.
Add ND6 stock solution to the culture medium at the final concentration of one microgram per microliter and incubate it to ubiquitously label endocytosed membrane compartments, such as endosomes. To reduce the extent of synaptic vesicles labeling, suppress the spontaneous neuronal activity pharmacologically. After selectively labeling synaptic vesicles as described in the manuscript, mount the cover slip containing cells in the imaging chamber.
Connect the temperature sensor, perfusion input, and vacuum tip to the imaging chamber. Adjust the perfusion speed to approximately 0.2 milliliters per second and start the perfusion of prewarmed normal tyrode's solution containing NBQX and DAP5 to remove excessive ND6 in the culture. Adjust the focus and locate the appropriate field of view containing healthy and well spread neurons bearing connected neurites.
Avoiding areas containing unresolved Dicolloids. Try imaging ND6 loaded cells with different exposure times to identify the best imaging settings. Set up the stimulation and perfusion protocol, frame interval, and total duration using the information in the text manuscript.
Start the image acquisition accompanied by synchronized stimulation and perfusion. Monitor the stimulation and solution exchange during imaging. Stop the perfusion after the imaging ends.
Remove the cover slip and clean the imaging chamber for the next trial. Back up and make an electronic copy of all image files. Choose and open the analysis program for data extraction.
Open or import an image stack to the analysis program. After generating a binary mask, use the analyze particle function with appropriate area size and circularity to solicit regions of interest, or ROIs, corresponding to cell membranes, endosomes, lysosomes, or synaptic boutons. Select four background ROIs in cell-free regions within the field of view.
Then save all selected ROIs. Align all frames within the ND6 time-lapse image stacks. Set the first image as the reference and align the rest to it using rigid registration, which will mitigate artifacts due to XY drifting.
Then save the aligned image stacks. Apply the saved ROIs to aligned ND6 image stacks. Measure the average pixel intensities of ND6 fluorescence for every ROI in every frame of the image stack.
Then export the results for statistical analysis. Calculate the mean intensity of the background ROIs to obtain the baseline noise, which will be subtracted from all selected ROIs. Average the three highest average intensities for every selected ROI during the application of pH 5.5 tyrode's solution to obtain the maximal fluorescence intensity, defined as 100%for normalization.
Average the three lowest average intensities for every selected ROI during the application of 50 millimolar ammonium chloride to set the minimal fluorescence intensity, defined as 0%for normalization. Calculate the relative fluorescence changes for every ROI based on its own 0%and 100%intensities. Derive mean fluorescence changes, change kinetics, and other values or plots from individual ROI data.
Bright green fluorescent puncta along the neuronal processes were visible after loading the neuronal cells with ND6. A strong correlation between ND6 and FM464 fluorescence intensities also suggested synaptic vesicle staining by ND6. A decrease was observed in the ND6 fluorescence in response to both electric stimulation and high potassium stimulation, suggesting that ND6 resides in the synaptic vesicle membrane and that the synaptic vesicle lumen is neutralized during the release.
Under an exhaustive electric stimulation, the M beta CD treatment significantly reduced synaptic vesicle release and retrieval measured by ND imaging, which suggested that membrane cholesterol facilitates synaptic vesicle exocytosis and endocytosis. Baf A1 prevented ND6 fluorescence recovery after stimulation by acutely blocking the reacidification of retrieved synaptic vesicles. Basic understanding of fluorescence microscopy is a must.
Careful planning for physiological conditions to support live cell imaging is very important. It is crucial to precisely determine the concentration of ND stock solution. If possible, remeasure stock solution concentration before every use.
Please keep it away from direct sunlight to avoid photo bleaching and probe decomposition.
