This method can help answer key questions in the protein trafficking field about nuclear and cytoplasmic translocation in system within which light imaging is not available. The main advantage of this technique is that it can be applied as a general protocol for the study of temporal protein movement without involving light imaging. 20 to 24 hours before the virus infection seed 5 times 10 to the 4 human embryonic lung or HEL fiber blast cells per well onto a four well 11 millimeter staggered slide and growth medium for overnight incubation at 37 degrees Celsius with five percent carbon dioxide.
It is important to rock the slide thoroughly to ensure an even distribution of the cells while taking care to avoid spilling the culture medium. The next day, replace the supernatants so the 70 to 80 percent confluent cultures with Medium 199, containing 410 plaque-forming units per cell, and place the slide at 37 degrees Celsius with shaking, for one hour. At the end of the incubation, replace the supernatants with fresh growth medium and return the cells to the incubator for the appropriate experimental infection period.
For immunofluorescent staining of the virus-infected cells, quickly wash the cells three times with PBS. Followed by an eight to ten-minute incubation in 200 microliters of four percent paraformaldehyde in PBS per well. At the end of the fixation, wash the cells three times with 200 microliters of PBS per wash, and permeabilize the cells with 100 microliters of 0.2 percent non-ionic surfactant per well for five to ten minutes.
Wash the permeabilized cells three times in PBS as demonstrated, and add 200 microliters of blocking buffer to each well for a one-hour incubation at room temperature. Next, add the appropriate concentration of the primary antibody of interest to each well for a two-hour room-temperature incubation. At the end of the incubation, remove any unbound primary antibody with three, ten-minute washes in fresh blocking buffer, and add an appropriate secondary antibody to each well for a one-hour incubation at room temperature, protected from light.
At the end of the incubation, wash the cells three times in a blocking buffer as demonstrated, followed by one wash in PBS and add one drop of antifade mounting medium supplemented with DAPI to each well. Then place a cover slip over the slide and seal the cover slope with transparent nail polish. Applying gentle pressure while mounting the cover slip will remove any excess mounting medium, helping to ensure helping to ensure the acquisition of high-quality images.
For confocal imaging of the labeled, infected cells, select the appropriate secondary antibody and DAPI wavelengths, and set the image format to 1, 024 by 1, 024 pixels with an average line of eight. Then image each well on the four-well slide under the 100x objective. For counting a large number of cells, obtain five to ten images from consecutive fields in the same well under the 40x objective.
To analyze the nuclear and cytoplasmic distribution of ICP0, open the project in the confocal application software and select an image. Open the Quantity tab, and select Sort Regions of Interest from the Tools menu. Select Draw Line, and draw a longitudinal line across the cell to be analyzed.
A histogram will appear showing the fluorescence intensity along the line for both the protein of interest, and DAPI. Based on the background staining, set up a constant threshold for the intensity of the protein of interest for analysis of the sub-cellular distribution of the protein in each experiment. If the protein signal, on average, is below the threshold in the nuclear region, but is above the threshold beyond the DAPI boundary, categorize the protein signal as predominantly located within the cytoplasm.
If the protein signal is above the threshold throughout the nucleus, and beyond the boundary of the DAPI signal, group the protein signal as a nucleus plus cytoplasmic localization. If the protein signal is above the threshold in the nucleus, but on average is below the threshold outside the boundary of the DAPI signal group the protein signal as a nuclear localization. Finally, tabulate more than 200 infected cells from each sample at different infection time points, and plot the data in a bar graph to illustrate the movement of the protein of interest over time.
As demonstrated, this method facilitates the analysis of the nuclear-to-cytoplasmic translocation of proteins of interest. For example, infected cell protein zero, or ICP0 subcellular distribution changes as a viral infection progresses. To understand the elements required for ICP0 trafficking during infection, ICP0 movements can be tracked in Wild Type and Mutant Type Herpes Simplex Virus 1-infected cells at different infection phases, allowing the evaluation of the sub-cellular distribution of ICP0 at different time-points of infection.
It is important to remember to use monolayer cultures so the virions have equal access to the cells and to normalize the multiplicity of infection according to the virus title so that infection progression is comparable between and within viruses. After each development, this technique paved the way for researchers in the fields of biology and cell virology to explore protein movement by studying the temporal subcellular localizations of proteins of interest within a cell population.
