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

1. Preparation of Bacteria and Host Cells

1. Transfer 12 or 14 mm glass cover slips in either 24- or 12-well plates, respectively, with sterile tweezers, counting one well per experimental condition. Sterilize these by UV treatment in a tissue culture hood for 15 min.
   NOTE: Include control wells for bacterial challenge and for SG induction by exogenous stresses.
2. For a 24-well plate with 12 mm coverslips, seed 1 x 10⁵ mammalian cells (e.g., HeLa cells) onto coverslips; press the coverslips down with a sterile pipette tip. Let cells adhere overnight at 37 °C in a 5% CO₂ tissue culture incubator in culture medium appropriate for each cell type.
   NOTE: Plate cells so that the confluence of cells is between 40 – 60% the following day.
   1. For HeLa cells, incubate in Dulbecco's Modified Eagle Medium (DMEM) with Non-Essential Amino Acids (NEAA) and 10% decomplemented Fetal Calf Serum (FCS). Decomplement FCS by heating for 30 min in a 56 °C water bath, swirling the serum once after 15 min.
3. To streak out bacteria, use a sterile pipette tip to remove a small amount from a bacterial glycerol stock (20% glycerol) stored in a -80 °C freezer and place it on a labeled plate. Use a sterile loop to spread the bacteria over about 10% of the plate. Use a new sterile loop and drag it through the smear to make 3 parallel lines, half a plate long. Streak through these lines covering the rest of the plate. Incubate plate O/N at 37 °C.
   1. Select a single bacterial colony (e.g., S. flexneri) from the freshly streaked-out plate. Avoid working with bacterial colonies older than 1 week.
   2. For S. flexneri strain M90T, inoculate a red colony from a freshly streaked out Tryptic Soy Agar-0.1% Congo red agar plate into 8 mL of Tryptic Soy broth in a 15 mL tube; tighten the lid and incubate shaking at 222 rpm O/N at 30 °C.
      CAUTION: Do not use large white colonies as they have lost the virulence plasmid and are non-infective.

2. Bacterial Challenge of Host Cells

1. Prepare bacteria to maximize infection potential.
   1. For S. flexneri, subculture bacteria by adding 150 µL O/N culture to 8 mL Tryptic Soy Agar and incubate shaking 222 rpm at 37 °C to late exponential phase (~2 h) to induce virulence gene expression and enhance infection.
      1. When the culture is at an optical density between 0.6 and 0.9 (measured at 600 nm), transfer 1 mL of culture into a 1.5 mL tube. Pellet bacteria for 2 min at 8,000 x g and resuspend in infection medium (DMEM with NEAA, lacking FCS) at OD₆₀₀ = 1. Thus, if the OD₆₀₀ is 0.85, resuspend in 850 µL.
         NOTE: For S. flexneri, at OD₆₀₀ = 1, there will be 8 x 10⁸ bacteria per mL when cultured in 8 mL medium. A Multiplicity Of Infection (MOI) of 20 is appropriate. For other pathogens, the number of bacteria per mL at an OD₆₀₀ and the appropriate MOI need to be determined empirically.
   2. To enhance bacterial-host interactions, coat bacteria with Poly-L-Lysine (PLL).
      NOTE: PLL treatment of S. flexneri had no effect on SG dynamics. Other pathogens need to be assessed for their amenability to PLL treatment.
      1. To coat bacteria with PLL, centrifuge 1 mL of bacterial culture for 2 min at 8,000 x g and then resuspend the pellet in 10 µg/mL PLL in Phosphate-Buffered Saline (PBS) at an OD₆₀₀ = 1.
      2. Add the bacterial solution to a small dish, such as a well within a 12-well plate, and incubate at RT (~ 20 °C) for 10 min with gentle agitation on a plate shaker.
      3. Pellet bacteria for 2 min at 8,000 x g, remove the excess PLL. Resuspend the pellet in 1 mL PBS and repeat this washing step twice. After the final wash, resuspend in infection medium at OD₆₀₀ = 1.
         NOTE: Here, the infection medium for S. flexneri is host cell medium with 20 mM HEPES w/o FCS.
2. Prepare cells for bacterial challenge.
   1. Remove medium from mammalian HeLa cells using a suction pump. Add 500 µL RT HeLa cell medium to wash the cells, swirl, remove medium using a suction pump, and replace with 500 µL RT appropriate infection medium (e.g., host cell medium with 20 mM HEPES w/o FCS).
   2. NOTE: For all washing steps, work quickly and process only up to 4 coverslips at a time to avoid drying out of the cells.
3. Challenge prepared HeLa cells with bacteria to infect 20 – 50% of the cells.
   NOTE: The appropriate time and MOI need to be determined for each bacterial species. Generally, a good start is 30 min at 37 °C with a MOI of 10.
   1. For S. flexneri, challenge HeLa cells cells using one of the two methods (step 2.3.1.1 or 2.3.1.2).
      1. Spin non-PLL-treated bacteria (20 µL of OD₆₀₀ = 1/well of 24-well plate in 500 µL medium) onto cells for 10 min at 13000 x g.
      2. Add PLL-coated bacteria (15 µL of OD₆₀₀ = 1/well of 24-well plate in 500 µL medium) for 15 min at RT to allow bacteria to settle onto cells.
   2. Allow the infection to occur by placing cells with settled bacteria into a 37 °C tissue culture incubator for 30 min.
      NOTE: For short time points, synchronize S. flexneri infection by placing plates in a 37 °C water bath for 15 min instead of in the tissue culture incubator.
4. Stop infection by washing cells.
   1. To wash cells and remove excess bacteria, remove the medium using a suction pump, add 1 mL fresh prewarmed (37 °C) HeLa culture medium (DMEM, 10% FCS, NEAA). Swirl the medium, remove and replace with fresh medium. Repeat twice and leave fresh medium on the cells.
      1. For S. flexneri, or other intracellular bacteria, after infection of HeLa cells and the washes, replace medium with standard tissue culture medium containing 50 µg/mL gentamicin to kill extracellular bacteria and prevent further cell invasion.
         NOTE: Do not add antibiotics in the medium for extracellular bacteria.
5. Incubate bacteria with cells for the desired amount of time in a tissue culture incubator.
   NOTE: For S. flexneri, infection may be as long as 6 h depending on the cell type. For HeLa cells, let infection proceed for 1.5 – 2 h before adding exogenous stresses to induce SG formation. For Caco-2 infections, in which Shigella spreads cell-to-cell, infect for 30 min to 6 h before adding exogenous stress. Cells may be fixed at this point without adding exogenous stresses to assess the formation of SGs as a result of the bacterial infection (in that case, proceed to section 4).

3. Inducing Stress Granule Formation by the Addition of Exogenous Stressors

1. Remove the medium from infected and non-infected HeLa cells using a suction pump, replace medium with 500 µL of appropriate medium with or without the stressor and incubate.
   NOTE: Stress-inducing conditions include the following (see below). For additional treatments see Kedersha et al.¹³.
   1. For heat shock treatment, use regular medium containing 20 mM HEPES and place the plate into 44 °C water bath for 30 min.
   2. For clotrimazole (induces mitochondrial stress) treatment, use regular medium without FCS with 20 µM clotrimazole and incubate in a tissue culture incubator for 1 h.
      NOTE: Store single-use aliquots of 20 mM clotrimazole stock in dimethyl sulfoxide (DMSO) at -20 °C for up to 4 months. Use DMSO vehicle for control cells.
   3. For arsenite (induces oxidative stress) treatment, use regular medium with 0.5 µM arsenite and incubate in tissue culture incubator for 1 h.
      NOTE: Store single-use aliquots of 0.5 mM arsenite stock at -20 °C for up to 6 months.
      CAUTION: Clotrimazole and arsenite are harmful and dangerous to the environment and must be disposed of appropriately.
   4. For Des-Methyl Des-Amino (DMDA)-pateamine treatment (inhibits eukaryotic translation initiation), use regular medium with 50 – 200 nM DMDA-pateamine and incubate in tissue culture incubator for 1 h.
      NOTE: Store DMDA-pateamine at -80 °C. Store reagents as small aliquots to avoid freeze thawing.

4. Fixation and Immunofluorescence Analysis of Stress Granule Formation

NOTE: Process the control and experimental coverslips at the same time to avoid staining differences that may impact image analysis in subsequent steps. The control samples include no infection with and without SG inducing treatment, and infected samples with and without SG inducing treatment.

1. Remove medium completely using a suction pump and fix samples by adding 0.5 mL RT 4% paraformaldehyde (PFA) in PBS. Incubate for 30 min at RT.
   CAUTION: PFA is harmful to the handler and the environment. Take proper measure to protect the handler and dispose with chemical waste.
   NOTE: Alternatively, fix for 15 min and then replace with -20 °C prechilled methanol for 10 min to retain more cytoplasmic localization of SG markers¹³.
2. Remove the PFA with a pipette and dispose the liquid in an appropriate waste container. Wash the coverslip with 1 mL Tris-Buffered Saline (TBS: 25 mM Tris, pH 7.3; 15 mM NaCl). Incubate the coverslip for 2 min with gentle shaking on a plate shaker during washing.
3. Remove TBS completely using a suction pump and add 500 µL 0.3% Triton-X100 in TBS for 10 min to permeabilize the cells.
4. Remove permeabilizing solution with a suction pump. Replace with 1 mL TBS, swirl the plate gently, remove TBS by suction, and add 1 mL blocking solution (5% FCS in TBS) for 1 h at RT.
5. Prepare the primary antibody solution (1:300 dilution) in 5% FCS in TBS. Calculate 50 µL per 12 mm coverslip and make enough for all coverslips in one batch.
   NOTE: The common primary antibodies that are used target G3BP1, eIF3b, and TIA1.
6. Spot 50 µL of the primary antibody mix onto a plastic paraffin film (parafilm) firmly taped onto the bench or chamber.
   NOTE: A list of canonical SG markers are provided in the Table of Materials, but composition of SGs may depend on the stress applied. Other SG markers are listed in Kedersha et al.¹³ Expected results for a number of SG markers for S. flexneri infection are listed in Table 1.
7. Pick up the coverslip with tweezers, dab the edge of the coverslip onto tissue, briefly releasing the tweezers to remove excess liquid, and gently place face down onto the drop. Incubate coverslips for 1.5 h at RT or overnight at 4 °C in a moist chamber.
   NOTE: To prepare a moist chamber, place prewetted tissues along the edges of a firmly closed chamber lined with plastic parafilm.
8. Transfer each coverslip with tweezers into a well of a new 24-well plate filled with 1 mL TBS; incubate with gentle shaking on a plate shaker for 5 min. Suction off TBS in each well using a suction pump, replace with 1 mL TBS and place back on plate shaker for 5 min. Repeat wash one more time.
   NOTE: Reuse the parafilm by removing the excess liquid with a tissue and washing the surface with water. Ensure the paraffin film is completely dry before using it for the subsequent staining step.
9. Prepare the secondary antibody solution in TBS (dilution 1:500 – 1:2,000). To stain the nucleus and bacteria, add 2 µg/mL DAPI to the mix. To stain actin, add fluorescent phalloidin. Pipette 50 µL secondary antibody mix onto the plastic paraffin film.
   NOTE: Use of highly purified cross-absorbed donkey secondary antibodies are recommended for co-localization studies of different SG markers when G3BP1 (goat antibody) is used. Choose fluorescently labeled secondary antibodies with non-overlapping spectra. eIF3b clearly stains the cytoplasm of cells and is therefore a good marker to delineate cells. The actin stain further helps to delineate cells at cell-to-cell contact sites and areas of bacterial entry.
10. Pick up the coverslip with tweezers, dab the edge of coverslip onto tissue, releasing briefly the tweezers, to remove excess liquid. Gently place coverslip face down onto the drop and incubate the coverslips in the dark for 1 h at RT.
11. Use tweezers to place the coverslip back into the 24-well plates filled with fresh 1 mL PBS. Ensure the coverslip is fully covered by the PBS. Wash the cells 3x with gentle shaking for 5 min each.
    NOTE: Keep the plate during the washings under a cover to protect from light as the fluorophores of the secondary antibody are light sensitive.
12. Briefly dip the coverslip into deionized water to remove the salt, dab dry from the edge onto a tissue and mount the coverslip onto 5 µL of fluorescence mounting medium on a glass slide. Seal the coverslip prior to imaging using nail polish. Keep slides at 4 °C for short-term (week) or at -20 °C for long-term (month) storage.
    NOTE: Avoid air bubbles when sealing as this will harm the image quality.

5. Fluorescence Imaging

NOTE: Refer to the user manual of the microscope for optimization of the set up.

1. Acquire images using a confocal microscope using either a 40 or 63X oil immersion lens. Select the desired fluorescent channels for image acquisition. When using multiple fluorescent channels, select sequential acquisition mode.
   NOTE: Start with the experimental samples where SGs have been induced the strongest to avoid overexposure on subsequent samples. Make sure to not have overexposed SGs throughout the entire depth of the cell.
   1. Set bit size of 12 or higher within the microscope settings to ensure accuracy of the image analysis.
   2. Set the image stacks to cover the whole depth of the cell. Check in the different channels and for different SG markers to ensure that the whole range is included. Set the beginning of the stack at the base of the cells and the top of the stacks at the height at the top of the cells where no more SG markers are observed.
   3. Acquire the stack. Keep the stack height the same for all images.
      NOTE: Do not change acquisition settings between control and experimental samples that will used for subsequent comparison.

6. Image Analysis

NOTE: Here, SG analysis on collapsed stacks using freeware ICY is described. Image analysis of 3D reconstructions may also be done using other specialized software. SG detection may be performed via a fully automated workflow established for this protocol (available at http://icy.bioimageanalysis.org/protocol/Stress_granule_detection_in_fluorescence_imaging). To use this protocol, the nuclei need to be stained with a DNA stain for the software to find the center of each cell, and the cell edges need to be marked by either a cytoplasmic marker (such as eIF3b) or actin stain for the software to identify the cell boundaries. The automatic workflow can be used to validate manually-derived results or directly for analysis when cell boundaries are detected with high confidence, which will mostly depend on the density of cells and the marker used to detect the cell boundaries.

1. Using ImageJ or Fiji, collapse the image stacks (Image>Stacks>Z projection), and save as .tiff files.
   NOTE: Create a new folder for each image as the image analysis software will link its results to the site of the original image.
2. Load a control and experimental .tiff image to the image analysis platform (File>Open).Go to the Sequence window. Scroll down in the "Lookup Table" to the channel parameters.
3. Deactivate all channels by clicking on the check box of all channel tabs. Click on channel 0 and then select the preferred color by clicking on the color bar above. Increase the intensity of the channel as needed to see all structures by clicking and moving the line in the intensity field. Repeat this for all channels.
   NOTE: Deactivate channels that are not required for a given task to minimize bias in sample analysis.
4. Scroll up and within the Canvas tab, zoom in to about 10 cells per field by adjusting the zoom tab.
5. Use the polygon function tools (in the top bar) to delineate cell boundaries of cells in the image. Use the actin stain or a cytosolic marker for cell boundary delineation (e.g. eIF3b).
   1. Click the polygon function within the "File & ROI" tools. Start delineation by clicking on the cell and then extend the line by making anchors through repeated clicking around the cell boundary. To finish a ROI, click again on the polygon function within the "File & ROI" tools.
      NOTE: Identify the sub-populations of the delineated cells that are infected. The sample consists of a mix of infected and non-infected cells. To identify bacteria, either use the DAPI stain or fluorescent bacteria (such as GFP-expressing bacteria). Increase the intensity to ensure that all bacteria are seen.
   2. To name a ROI, go to the ROI window on the right and click on the cell of interest in the image. This will highlight the corresponding ROI in the ROI tab. Double click on the ROI name and modify the name (e.g. infected cell #1).
      NOTE: If one image is to be used to analyze both infected and non-infected cells, it is easier to save the image in two separate folders and delineate the infected and non-infected cells separately, generating two different spreadsheets, which will facilitate analysis later.
6. Open the spot detector application within the "Detection & Tracking" tab (top bar). Within the Input tab, the current image will be selected. Do not change anything. Within the Pre-Processing tab, select the channel to be analyzed.
   NOTE: Only one channel can be analyzed at any given time. A batch analysis can be selected here if one uses the fully automated analysis sequence whose parameters have been checked to give satisfying results.
   1. Within the Detector tab, chose "Detect bright spots over dark background". Then select the appropriate scale and sensitivity. Do this by testing different settings and cross-checking spot detection in both the control and the experimental samples.
      NOTE: For an image with a 2,048 x 2,048 resolution and a pixel size of 0.12 µm², a level 2 threshold with a sensitivity of 25 to 100 is generally appropriate but each SG marker has to be tested empirically. Set up spot detection so that in the non-treated control sample, spots are not detected, while in the experimental sample with SGs, all the small and large SGs are counted.
   2. Within the ROI tab, select the suggested ROI From Sequence. Within the Filtering tab, select No Filtering. In the Output tab, choose the appropriate output.
      NOTE: Choosing Enable Specific file is helpful for saving different detection conditions or different channels. Selecting to export the binary image and the original image with ROIs and detection is helpful for quality control analysis.
   3. Select "Remove previous spots rendered as ROIs". Choose the folder to save the file by clicking on the "no file selected" button. Within the Display tab, select the desired options.Click "Start detection".
      NOTE: A folder with the name and the location chosen will be created that contains the exported imaging options. These images will be overwritten for each new condition tested. To keep the images, either rename the folder so that a new folder will be created or transfer the images from the save folder to a new folder. For quality control of the images, it is helpful to select display detection mark, number of detection of ROI and ROI number and name.
7. Consolidate and save the data for the different parameters using the generated spreadsheet for each image or condition tested.
   NOTE: The parameters to analyze include the number of SGs per ROI, the SG surface areas, and the SG maximum, average and minimum intensities.
8. Transfer the data and analyze using an analysis program capable of analyzing, graphing and determining the statistical significance.