Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

The overall goal of this experiment is to assess the effects of a bacterial infection on the ability of host cells to form stress granules to response to exogenous stress. This method is a valuable tool in the field of cellular microbiology for assessing whether or not a given pathogen is able to alter or subvert the whole stress granule pathway. The main advantage of this technique is that stress granules can be qualitatively and quantitatively be analyzed in a rigorous and automated manner using free image analysis programs.

This methods provides insight into the dynamics of stress granule formation and composition and how these parameters are affected by a specific pathogen. Thank you. Before beginning the challenge, inoculate a red S.flexneri M90T bacteria colony from a freshly streaked tryptic soy agar 1%Congo red agar plate into a 15 milliliter conical tube, containing eight milliliters of tryptic soy broth.

After tightening the lid, incubate the colony overnight with shaking at 222 RPM and 30 degrees Celsius. The next day, subculture 150 microliters of the bacteria in eight milliliters of fresh tryptic soy broth for about two hours at 37 degrees Celsius and 222 RPM to initiate a late exponential phase induction of virulence gene expression and to enhance the bacterial infectability. When the optical density of the subculture is between 0.6 and 0.9, transfer one milliliter of the bacteria into a 1.5 milliliter tube for their collection by centrifugation.

And re-suspend the pellet in infection medium at an optical density of one. Next, aspirate the supernatants from each well of a HeLa cell coverslip culture. And carefully wash the HeLa cells with 500 microliters of fresh room temperature HeLa cell medium per well.

Replace the wash with 500 microliters of infection medium per well, and centrifuge the 24 well plate to spin the bacteria onto the cells. Transfer the settled bacterial cocultures into a 37 degree Celsius tissue culture incubator for 30 minutes to facilitate the host cell infection. At the end of the incubation, remove the exogenous bacteria from the cells with three rinses in one milliliter of fresh 37 degree Celsius HeLa culture medium per wash.

Then cover the infected cells with one milliliter of fresh culture medium containing gentamicin and return the plate to the cell culture incubator. At the end of the incubation, replace the medium with 500 microliters of the appropriate culture medium, supplemented with the stressor of interest, and return the cells to the tissue culture incubator. After one hour, aspirate the supernatant completely, and fix the samples in 0.5 milliliters of room temperature 4%paraformaldehyde in PBS for 30 minutes.

Next, discard the fixative into the appropriate waste container, and wash the coverslips with one milliliter of tris-buffered saline for two minutes with gentle shaking. At the end of the wash, completely aspirate the TBS and permeabilize the cells in each well with 500 microliters of 0.3%Triton X-100 in TBS. After 10 minutes, replace the permeabilizing solution with one milliliter of TBS, swirl the plate gently, and replace the TBS with one milliliter of blocking solution for one hour at room temperature.

To label the cells with a primary antibody cocktail of interest, spot 50 microliters of the primary antibody mix per coverslip onto a piece of plastic Parafilm firmly taped onto the bench top. Then use tweezers to pick up the first coverslip. And dab the edge of the coverslip onto a piece of tissue paper to remove the excess liquid.

Carefully place the coverslip onto the drop and incubate the coverslips under the appropriate labeling conditions. At the end of the primary antibody labeling incubation, transfer each coverslip into an individual well of a new 24 well plate containing one milliliter of TBS per well, and wash the cells at room temperature on a plate shaker for five minutes three times. After the last wash, label the cells on each coverslip with the appropriate secondary antibody cocktail mix, as just demonstrated.

And incubate the coverslips in the dark for one hour at room temperature. At the end of the secondary antibody labeling incubation, wash the cells in one milliliter of PBS three times in a new 24 well plate with gentle shaking as just demonstrated, but protected from light. After the last wash, briefly dip each coverslip in deionized water to remove the salt, dab the coverslip edges onto a tissue, and carefully mount the coverslips onto five microliters of fluorescence mounting medium per glass microscope slide without bubbles.

Then seal the coverslip with nail polish. And store the slides as appropriate until imaging. To image the cells by fluorescence confocal microscopy, begin by selecting the appropriate objective and the appropriate fluorescent channels for image acquisition.

Then acquire image stacks of the cells, using the appropriate imaging software. When all of the images have been captured, use the appropriate imaging analysis software to collapse the image stacks and save the stacks as TIFF files. Next, load a control and an experimental TIFF image to the image analysis platform, and select Lookup Table to open the channel parameters in the Sequence window.

Click on all of the channel tab checkboxes to deactivate all of the channels. Then click channel zero, and click on the channel zero color bar to select the preferred color, increasing the intensity of the channel as necessary to visualize all of the structures. After selecting and adjusting each of the appropriate color channels for the experimental analysis, select the canvas tab and adjust the zoom tab to visualize about 10 cells per viewing field.

Using the polygon tool, click on the cell of interest and extend the line around the cell to delineate the cell boundary. To name this region of interest, in the region of interest window, right-click on the cell of interest, and double-click on the region of interest's name to modify it. After selecting and naming all of the cells of interest in the same manner, open the Spot Detector application within the detection and tracking tab.

Open the Pre Processing tab and select the channel to be analyzed. Within the Detector tab, select Detect bright spots over dark background and select the appropriate scale and sensitivity. Under the Region of Interest tab, select the suggested region of interest from the sequence.

Under the Filtering tab, select NoFiltering. In the Output tab, select the appropriate output. Select Remove previous spots rendered as regions of interest and click no file selected to select the folder for saving the file.

Then under the Display tab, select the appropriate options of interest and click Start Detection. Within the imaging analysis software, the spot detector can be used to identify the stress granules within each of the designated regions of interest. A binary image can then be generated to display all of the identified stress granules.

Stress granule analysis must be carefully tailored to each stress granule market analyzed. For example, changing the size requirement of the region of interest detected, or changing the sensitivity of the detection parameters lead to varying results. At higher pixel size scales, smaller stress granules will not be counted, and the numbers of stress granules will decrease.

While increasing the sensitivity results in an increase in the number of stress granules. For example, in this experiment, a scale of two with a sensitivity of 100 gave the best result from the stress granule marker G3BP1. While a scale of two with a sensitivity of 55 gave the best result for the EIF3B stress granule marker.

The surface area can then be calculated from the size of the stress granules. Frequency distribution plots are useful for highlighting shifts in the size of stress granules between different cell populations. In addition, the intensity of the fluorescence can provide information about the quality of the stress granules analyzed.

Once mastered, the whole cell challenge can be done in about six to seven hours, and the analysis can be completed in another two to three hours. While attempting this procedure, it's important to remember to always process the control and the experimental samples at the same time and under the same conditions to minimize variability within the data. Following this procedure, the analysis can also be extended to a three-dimensional volume to get additional insights into stress granule localization.

This semi-automated procedure allows for a rigorous and unbiased analysis of stress granules in uninfected and infected cells. It can reveal previously unknown subversions of the stress granule pathway. After watching this video, you should have a good understanding of how to infect cells with bacteria, how to induce stress granule formation, and how to use a semi-automated approach for analyzing stress granules in a qualitative and quantitative manner.

And don't forget that with Shigella flexneri and stress granule inducing agents like clotrimazole can be hazardous, and that precautions such as wearing gloves, working in a BL2 lab, and properly discarding all of the reagents are necessary.