Our protocol can be used to investigate the motility and localization of astrocytic organelles or proteins under controlled extracellular environments and mild disease states. Unlike the MHA fixed preparations, which provide single snapshots of protein and organelle localization, our live imaging technique can be used to analyze particle dynamics in individual live astrocytes. The day before the transfection seed astrocytes at the appropriate density for imaging at 24 to 48 hours post transfection.
Dilute the lipofection reagent in reduced serum medium to the appropriate experimental concentration. Dilute five micrograms of high purity DNA in 250 microliters of reduced serum medium and add five microliters of lipofection enhancer reagent to the tube. Mix the lipofection reagent with an equal volume of the DNA lipofection enhancer mix and incubate the solution for 15 minutes at room temperature.
At the end of the incubation, remove the supernatant from the astrocyte culture and add 100 microliters of the transfection mix dropwise to the cells. After six hours at 37 degrees Celsius and 5%carbon dioxide, replace the transfection complex with an appropriate volume of astrocyte culture medium and return the cells to the cell culture incubator for an additional 24 to 72 hours. 30 minutes before the imaging, dilute a suitable lysosomal labeling probe in 200 microliters of astrocyte culture medium to a working concentration of one micromolar and label the astrocytes with a probe for 30 minutes at 37 degrees Celsius.
Then wash the cells one time with warm astrocyte culture medium and replace the wash with imaging medium. Immediately after the probe labeling, place the astrocyte culture container into the appropriate adapter on the microscope stage and using EBI fluorescence light, locate the cells that express fluorescent proteins or probe. Use the digital camera to adjust the fluorescent sample illumination to visualize the selected cells and adjust the focus and zoom in to a single cell.
Then use the Zoom and Definite Focus functions to acquire a single Z-stack time-lapse series at a frequency of one frame every two seconds for time intervals ranging between 300 and 500 seconds. Save and export the time-lapse images as AVI or TIFF stack files. To analyze the time-lapse images, open the time-lapse image sequence in ImageJ or Fiji and use the Split Channel tool to split the channels.
In the eight bit green channel image, use the Segmented Line tool to trace a line along the trajectories of the particles using the same desired directional convention for all of the movies so that the polarity is consistent within and across all of the cells being studied. Double click the Line tool to adjust the line width to match the thickness of the track and run the Draw Kymo macro of the Kymo ToolBox plugin using a line width of 10. A prompt asking to calibrate the image in time and space will appear.
Once calibrated, kymographs will be generated and can be saved as TIFF files. To assign particle trajectories, use the Segmented Line tool to manually track each particle in the kymograph for the full duration of the acquisition and use the ROI manager to record each particle trajectory as a region of interest. Save all of the regions of interest per each time-lapse video for further analysis and run the Analyze Kymo macro of the Kymo ToolBox plugin.
A window will open asking to define the outward direction of particle motion from the nucleus of the cell of interest from the dropdown menu. The limit speed should be defined according to the sensitivity of the software for each cargo of interest and the line width should be adjusted to match the thickness of each particle trajectory. The Log All Data and Log Extrapolated Coordinates should be for calculation of the various cargo transport parameters.
Click Okay. The data calculated for the individual tracks will then be pooled per kymograph and saved in text files specific to each image. Combining cytosine arabinoside treatment with the shaking based purification strategy enriches the purity of the astrocyte cultures over traditional protocols that include the purification step only.
Lipofection based transfection allows the transient expression of proteins at levels that are optimal for live cell imaging without causing toxicity or affecting astrocyte viability. Similarly, the use of a fluorescent probe permits the rapid and efficient labeling of acidic endolysosomal organelles for the tracking of organelle dynamics and astrocytes. The time-lapse data from the imaging can be used to generate kymographs that track cargo motion in time and space.
In these kymographs, anterograde movement of the indicated cargo is represented by trajectories with negative slopes, while retrograde movement is represented by trajectories with positive slopes. Stationary vesicles appear as vertical trajectories. In this analysis, quantification of the flux of a specific cargo through an area of the astrocyte revealed differences in the percentage of modal particles among cargoes that are likely representative of their normal base line motility in the region of the astrocyte within which the movies were acquired.
The precise mapping of the change in the X, Y position along the full timescale for each particle obtained from the kymograph can also be used to evaluate other motion parameters, such as cargo velocity and run length. As this protocol requires high quality astrocyte cultures and efficient cargo labeling, the transfection reagent incubation time and acquisition timeline should be adjusted for each plasmid or cargo of interest. This protocol can be modified to characterize transport events in astrocytes in response to cell damage, toxicity, pathogenic mutations, synaptic activity, or changes in the intra or extracellular environment.
