1. Bacterial Culture

1. Generate isogenic mutant strains to be used for the study of the specific secreted factor of interest²¹.
   Note: The GASM1T1 5448 strains utilized for these experiments included WT GAS', a sagAΔcat SLS-deficient mutant (abbreviated in text as ΔsagA), and a sagA complemented strain (abbreviated in text as ΔsagA+sagA)²¹.
2. Prepare overnight liquid cultures of bacterial strains of interest.  Grow GAS M1T1 5448 strains in  -10 ml of Todd-Hewitt broth at 37 °C for 16-20 hr prior to infecting human cells.
3. Centrifuge overnight bacterial cultures (2,400 rcf for 10 min) and resuspend in fresh bacterial medium.
   Note: Resuspension in the same volume as the overnight cultures were grown in (5-10 ml) generally produces a desirable initial optical density.
4. Normalize bacterial cultures to equal starting concentrations by diluting the re-suspended cultures in additional bacterial medium until the same optical density at 600 nm (OD₆₀₀) is obtained for all strains to be tested (OD₆₀₀ of approximately 1.0 is recommended for convenience).
   Note: In these studies, stationary phase bacterial cultures were used to infect host cells immediately following normalization. However, as some strains of bacteria exit stationary phase nonsynchronously it may be more appropriate to begin host infections with log phase cultures if this is found to be the case for the strains of interest.
5. Prior to further analysis, perform a colony counting assay to determine the colony forming units (CFU) per milliliter present in the starting cultures so that the multiplicity of infection (MOI), or ratio of bacteria per host cell, can be determined.
   1. Prepare 1 ml serial dilutions of bacterial cultures that have been normalized to the desired optical density in phosphate buffered saline (PBS) or appropriate bacterial growth medium (Todd-Hewitt broth was used in these studies). Prepare dilutions ranging from 10^-1 to 10^-6 to produce at least one set of agar plates with countable colonies (30-300 colonies). Perform all dilutions in triplicate.
   2. Transfer 100 µl of each serial dilution onto an agar plate containing appropriate medium for the bacterial species being analyzed.
      Note: Todd-Hewitt agar was used in these studies.
   3. Use a glass or plastic bacterial spreader to evenly distribute the 100 µl aliquot across the entire surface of the agar plate, and continue spreading until the liquid has been absorbed into the agar. Perform under flame using sterile technique.
   4. Incubate the agar plates at 37 °C overnight or until countable colonies appear.
   5. Count the colonies that grow on each plate and calculate the CFU/ml in the original culture by the following formula: number of colonies x inverse of the serial dilution / culture volume plated in milliliters.
      e.g. (200 colonies x 1/10^-5 dilution) / 0.1 ml plated = 2.0 x 10⁸ CFU/ml

2. Host Cell Culture

1. Obtain an appropriate host cell line for the desired analyses.
   Note: HaCaT human epithelial keratinocytes²⁹, a kind gift from V. Nizet, were used for these studies.
   1. Maintain HaCaT cells in 100 mm culture dishes in Dulbecco's Modified Eagle's Medium (DMEM) with 10% heat inactivated fetal bovine serum (FBS). Incubate at 37 °C with 5% CO₂.

3. Host Cell Infection with Permeable Membrane Insert System

1. Plate cells in appropriate tissue culture dishes and allow them to grow until they reach 90% confluency.
   Note: The HaCaT cells used in these studies typically reach confluency within 2-3 days after plating.
   1. For lysate collection (e.g. signaling analysis and adenosine triphosphate (ATP) determination assays), plate HaCaT cells in 6 well dishes at a seeding density of approximately 3 x 10⁵ cells/well.
   2. For immunofluorescence imaging experiments, plate cells on sterile glass coverslips within 6 well dishes at a seeding density of approximately 3 x 10⁵ cells/well.
   3. For ethidium homodimer membrane permeabilization and lactate dehydrogenase (LDH) release assays, plate HaCaT cells in 24 well dishes at a seeding density of approximately 5 x 10⁴ cells/well.
2. Immediately prior to treatment, wash cells with 1x sterile PBS.
3. Apply fresh growth medium to cells. For the conditions described here, apply 2 ml medium per well for 6 well plates or 0.5 ml medium per well for 24 well plates. If pharmacological treatments are being tested, mix with fresh medium and apply during this step.
   Note: Some assays may require alternative media. For example, as phenol red and high serum concentrations interfere with the LDH release assay, phenol red-free DMEM supplemented with 1% bovine serum albumin (BSA) (w/v), 2 mM L-glutamine, and 1 mM sodium pyruvate was utilized for these experiments.
4. After fresh medium has been applied, carefully place a sterile 0.4 µm permeable membrane insert in each well. Perform this step in a laminar flow hood, and use sterile forceps to transfer the permeable membrane insert into each well.
5. Apply fresh cell culture medium to the upper chamber of each well according to the manufacturer's instructions. For the conditions described here, apply 1 ml medium per well for 6 well plates or 0.1 ml medium per well for 24 well plates.
6. Apply an appropriate volume of normalized bacterial cultures (see section 1) to the upper chamber of the permeable membrane insert system. Use bacterial medium for uninfected control treatments. For the results described here, add 25 µl of the normalized cultures per chamber for 24 well plates and add 100 µl of the cultures per chamber for 6 well plates.
   Note: In these studies, wild-type or mutant GAS was applied to host cells at a MOI of 10. MOI was calculated based on the final eukaryotic cell numbers (e.g. 2 x 10⁶ cells/well), such that 10 times as many bacteria were added per host cell at the beginning of the infection period (e.g. 2 x 10⁷ CFU per well). Appropriate MOI will vary with different bacterial species and with the desired follow-up analyses.
   Note: In addition to using bacterial medium for uninfected controls, other useful controls for certain applications of this technique may include heat-killed bacteria or spent bacterial medium for comparison.
7. Incubate infected cells at 37 °C with 5% CO₂.

4. Sample Collection

1. To collect cell culture medium, host cell lysates, or glass coverslips with intact cells for follow-up analyses, begin by carefully removing the permeable membrane insert with sterile forceps.
2. For Assessment of Secreted Host Protiens:
   1. Collect the medium from the lower chamber into 1.5 ml tubes. Avoid disturbing the monolayer during collection.
   2. Centrifuge samples at 14,000 rcf for 10 min at 4 °C to remove cellular debris.
   3. Remove all but 50 µl of the supernatant to a fresh 1.5 ml tube and store at -20 °C or use immediately.
3. For Assessment of Host Cell Lystates:
   1. Gently aspirate the medium above the monolayer of host cells. Avoid disturbing the monolayer, as this may result in sample loss.
   2. Rinse cells once with 1x PBS.
   3. Gently aspirate PBS. Immediately apply an appropriate volume of ice-cold lysis buffer to achieve the desired protein concentration, and incubate the samples on ice for 15 min. Apply between 200 µl and 350 µl of lysis buffer per well of each 6 well plate to achieve protein concentrations between 0.5 and 1.5 mg/ml.
      Note: The lysis buffer used in these studies was composed of deionized water, 1% (v/v) Nonidet P40 Substitute, a phosphatase inhibitor cocktail, and a protease inhibitor cocktail. Specific reagents are listed in the Materials and Equipment section.
   4. Use a cell scraper to detach the cells from the plate surface of each well and pipette the entire contents of each well into a 1.5 ml tube.
   5. Centrifuge samples at 14,000 rcf for 20 min at 4 ˚C.
   6. To assess soluble lysate components, remove the supernatant to a fresh tube and store at -20 ˚C or use immediately.
   7. To assess nuclear or other insoluble lysate components, reserve the pellet and store at -20 ˚C or use immediately.
4. For Assessment by Immunofluorescence Imaging:
   1. Aspirate medium and wash cells in 1x cold PBS.
   2. Fix cells overnight in 4% paraformaldehyde (PFA) solution in PBS (w/v).
      Note: In these studies, 1 ml of PFA was added per well of each 6 well plate.

5. Suggested Applications

1. Host Signal Transduction Analysis by SDS-Page and Western Blotting
   1. Collect host cell lysates as described in section 4.3.
   2. Determine the protein concentration of each sample lysate by bicinchoninic acid (BCA) assay or equivalent using protein standards³⁰.
   3. Normalize protein concentrations between samples prior to loading and running the samples on a 4-15% polyacrylamide gel or appropriate alternative³¹.
      1. Normalize sample concentrations by pipetting the same protein amount from each sample (e.g. 20 µg) into a new 1.5 ml tube and adding variable volumes of lysis buffer (buffer volume = total volume – sample volume added) to each tube to make the total volume (e.g. 50 µl) and final protein concentration equivalent across samples.
   4. Transfer samples to a polyvinylidene fluoride (PVDF) membrane (or equivalent). For the results described here, transfer samples at 25 volts for 2 hr before increasing the voltage to 70 volts for an additional 45 min. Use transfer buffer composed of 200 mM Tris base, 1.5 M glycine and 20% methanol (v/v) in deionized water.
   5. Block membranes in 5% BSA + 0.1% Tween 20 in Tris buffered saline (TBS). Adjust accordingly based on manufacturer recommendations for the antibody to be used.
   6. Incubate membranes with primary antibodies overnight at 4 °C. Use at manufacturer recommended dilution.
   7. Wash membranes for 1.5 hr in TBS+0.1% Tween 20; refresh buffer every 10-15 min.
   8. Incubate with Horseradish Peroxidase (HRP)-conjugated secondary antibody at a dilution of 1:5,000 for 1.5 hr at room temperature.
   9. Wash membranes for 1.5 hr in TBS + 0.1% Tween 20; refresh buffer every 10 – 15 min.
   10. Incubate membranes with chemiluminescence detection reagent prior to developing on film according to the manufacturer's instructions. Other detection methods may be used as desired.
2. Host Cell Immunofluorescence Staining and Imaging
   1. Fix coverslips containing host cells as described in section 4.4.
   2. Remove PFA solution and wash coverslips containing treated cells two times in PBS, aspirating between washes.
      Note: PFA is highly toxic and must be disposed of appropriately.
   3. Block the coverslips for 2 hr at room temperature in PBS with 1% (w/v) normal goat serum, 2% (v/v) Triton X-100, and 0.5% (v/v) Tween 20.
   4. Wash coverslips with PBS for 30 min, refreshing the buffer every 10 min.
   5. Incubate the coverslips with primary antibody at a 1:50 ratio (or as recommended by manufacturer) in blocking solution overnight at 4 °C.
   6. Wash the coverslips for 1.5 hr in PBS, refreshing the buffer every 30 min.
   7. Incubate the coverslips for 2 hr at room temperature in secondary antibody (Goat anti-rabbit IgG AlexaFluor488 was used for these studies) using a 1:200 ratio of antibody to blocking solution.
   8. Wash coverslips for 1 hr, refreshing the buffer every 20 min.
   9. Apply desired stains.
      Note: These studies utilized DAPI (nuclear stain) and rhodamine-phalloidin (actin stain) at ratios of 1:1,000 in blocking solution.
   10. Incubate coverslips with nuclear and actin stains for 30 min at room temperature.
   11. Wash coverslips in PBS for 30 min at room temperature, refreshing the buffer every 10 min.
   12. Mount the coverslips on glass slides using mounting medium.
   13. Allow coverslips to set on the slides overnight prior to sealing and imaging. For the results described here, collect images on a fluorescence microscope using a standard 20X objective. Use ImageJ and microscope imaging software to process the captured images.
3. Ethidium Homodimer Cell Death Assay
   1. Aspirate medium from each well and wash cells twice with sterile PBS.
   2. Apply 4 µM ethidium homodimer-1 in PBS.
   3. Incubate cells at room temperature for 30 min in the dark.
   4. Determine the level of fluorescence using a plate reader set to 528 nm excitation and 617 nm emission with a cutoff value of 590 nm.
   5. Add 0.1% (w/v) Saponin to each well following the initial reading and allow the plate to incubate for an additional 20 min at room temperature (with rocking) before reading the plate a second time at the same settings.
   6. Determine percent membrane permeabilization individually for each well by dividing the initial fluorescence reading (post-treatment) by the second fluorescence reading (post-Saponin).
4. ATP Determination Assay
   1. Collect lysates as described in section 4.3 above.
   2. Normalize protein levels in the lysates via BCA assay or similar³⁰.
   3. Determine cellular ATP levels using a luminescence-based ATP determination kit or equivalent according to the manufacturer's instructions.
   4. Normalize treated values to the corresponding uninfected control samples.
5. LDH Release Assay
   1. Following infection, collect supernatants as described in section 4.3.
   2. Evaluate LDH release using a cytotoxicity detection kit for LDH release according to the manufacturer's instructions.