Quantitative Time-Lapse Microscopy for Assessing Extracellular Matrix Stiffness-Dependent Bacterial Dissemination

1. Manufacturing Thin Two-layered Polyacrylamide (PA) Hydrogels on Multi-Well Plates

1. Dissolve ammonium persulfate (APS) in distilled ultrapure water to achieve a final concentration of 10 g/mL. Aliquot and store the solution at 4 °C for short-term use (3 weeks).     
   NOTE: The above solution can be prepared in advance of hydrogel fabrication.
2. Glass activation of 24-well dishes
   1. Incubate 24-well glass bottom plates with 13 mm-diameter wells (see Table of Materials) for 1 h with 500 µL of 2 M sodium hydroxide (NaOH) per well at room temperature.
   2. Rinse the wells 1x with ultrapure water and then add 500 µL of 2% (3-Aminopropyl) triethoxysilane (see Table of Materials) in 95% ethanol to each well for 5 min.
   3. Rinse the wells 1x again with water and add 500 µL of 0.5% glutaraldehyde to each well for 30 min. Rinse the wells 1x with water and dry them at 60 ˚C with the lid off.
3. Polyacrylamide hydrogel fabrication
   NOTE: See Figure 1.
   1. Prepare aqueous solutions that contain 3 – 10% of 40% stock acrylamide solution (see Table of Materials) and 0.06 – 0.6% of 2% stock bis-acrylamide solution (see Table of Materials) to manufacture hydrogels of tunable stiffness ranging from 0.6 kPa to 70 kPa. See Table 1.
      1. For 0.6 kPa hydrogels, mix 3% acrylamide with 0.045% bis-acrylamide. For 3 kPa hydrogels, mix 5% acrylamide with 0.074% bis-acrylamide. For 10 kPa hydrogels, mix 10% acrylamide with 0.075% bis-acrylamide. For 20 kPa hydrogels, mix 8% acrylamide with 0.195% bis-acrylamide. For 70 kPa hydrogels, mix 10% acrylamide with 0.45% bis-acrylamide.  
         NOTE: Further details on achieving the desirable PA hydrogel stiffness can be found elsewhere.
   2. Prepare two aqueous solutions for each desirable hydrogel stiffness. Prepare Solution 1 to be bead-free and Solution 2 to contain 0.03% 0.1-µm diameter fluorescent micro-beads (see Table of Materials).
   3. Degas Solutions 1 and 2 by vacuum for 15 min to eliminate oxygen that is known to inhibit polymerization of the solutions.
   4. Add 0.6% of the 10 g/mL stock APS solution and 0.43% tetramethylethylenediamine (TEMED) to Solution 1 to enable a polymerization initiation. Act fast.
   5. Add 3.6 µL of Solution 1 to the center of each well of the 24-well dish (see step 1.2 for its preparation).
   6. Immediately cover the wells with 12-mm untreated circular coverslips and let Solution 1 sit for 20 min so that it fully polymerizes.
   7. Gently tap a syringe needle to a surface to create a small hook at its tip to facilitate the removal of the coverslips. Lift the coverslips using the syringe needle.
   8. Add 0.6% of the 10 g/mL stock APS solution and 0.43% TEMED to Solution 2. Deposit 2.4 µL of the mixture on top of the first layer in each well of the 24-well dish.
   9. Cover Solution 2 with 12-mm circular glass coverslips, gently pressing downwards using a pair of forceps to ensure the thickness of the second layer is minimal. Let Solution 2 polymerize for 20 min.
   10. Add 500 µL of 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5 to each of the wells and then remove the glass coverslips with the syringe needle and forceps.
4. Sterilization, collagen-coating, and equilibration of polyacrylamide hydrogels
   1. UV-expose the hydrogels for 1 h in the tissue culture hood to allow sterilization.
   2. Prepare a mixture of 0.5% weight/volume sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylamino)hexanoate (Sulfo-SANPAH, see Table of Materials) in 1% dimethyl sulfoxide (DMSO) and 50 mM HEPES pH = 7.5.
   3. Add 200 µL of this solution to the upper surface of the hydrogels. Acting fast, expose them to UV (302 nm) for 10 min to activate them.
   4. Wash the hydrogels twice with 1 mL of 50 mM HEPES pH = 7.5. Repeat if needed to ensure that any excess crosslinker is removed.
   5. Protein-coat the hydrogels with 200 µL of 0.25 mg/mL rat tail Collagen I (see Table of Materials) in 50 mM of HEPES. Incubate the hydrogels, with the collagen solution on top, overnight at room temperature.
      NOTE: To prevent dehydration/evaporation, place the multi-well plates in a secondary containment and add laboratory cleaning tissues soaked in water in the inner periphery of the containment.
   6. Use an epifluorescence or confocal microscope with a 40X objective to measure the thickness of the hydrogels. Do so by locating the z positions of the bottom (where the glass surface is) and top planes of the hydrogel (where the fluorescent beads' intensity is maximum). Then subtract the z positions to determine the height.   
      NOTE: We used an inverted epifluorescence microscope and a 40X objective with numerical aperture 0.65 to measure the thickness of the hydrogels. Atomic Force Microscopy (AFM) measurements can also be performed at this point to confirm the exact stiffness of the hydrogels (see Figure 2).
   7. Before seeding the cells of interest on the hydrogels, add 1 mL of media on the hydrogels and incubate them at 37 ˚C for 30 min to 1 h to ensure equilibration.
      NOTE: We added MCDB-131 full media because that is the media where the model host cells (HMEC-1) were cultured in (see step 2.1 for details).

2. Human Microvascular Endothelial Cell Culture and Seeding on Hydrogels

1. Culture HMEC-1 (human, microvascular endothelial) cells in MCDB-131 full media containing MCDB-131 media supplemented with 10% fetal bovine serum, 10 ng/mL of epidermal growth factor, 1 µg/mL of hydrocortisone, and 2 mM of L-glutamine (see Table of Materials).
2. Split the confluent cultures 1:6 every 3-4 days and keep the cells until passage 40.
3. One day prior to the experiment, detach the cells from their culture vessel using 0.25% trypsin/EDTA. First, wash the cells and their culture vessel 1x with sterile phosphate buffered saline (PBS), and then add the appropriate amount of 0.25% trypsin/EDTA (2 mL per 100-mm dish or 75-cm flask, 1 mL per 60-mm dish or 25-cm flask), incubating the flask at 37 °C for 5 – 10 min to allow the detachment of the cells from their substrate.
4. Neutralize the trypsin by adding the desired volume of MCDB-131 full media, pipet gently to break up the clumps of cells, and then place the solution into a conical centrifuge tube.
5. Gently swirl the solution of cells to ensure that the cells are evenly distributed and then take out 20 µL of the solution and very gently fill out the two chambers underneath the coverslip of a glass hemocytometer.
6. Pellet down the solution of cells contained in the conical centrifuge tube using centrifugation for 10 min at 500 x g.
7. During the 10 min waiting period, count the cells using a hemocytometer. Use a microscope, focus on the grid lines of the hemocytometer with a 10X objective, and then use a hand tally counter to count the number of cells in one 1 mm x 1 mm square.
8. Move the hemocytometer to another 1 mm x 1 mm square, count the cells there and then repeat the process two more times. Calculate the average of the four measurements and then multiply the average by 10⁴. The final value is the number of viable cells/mL in the cell suspension that is being centrifuged.
9. Remove the liquid out of the conical centrifuge tube while ensuring that the cell pellet is not disrupted. Resuspend the cells in the MCDB-131 full media at a concentration of 4 x 10⁵ cells/mL.
10. Seed the cells in suspension on the hydrogels by first removing the media with which the hydrogels were incubated and then adding 1 mL of cell suspension on each hydrogel.

3. Infection of Human Microvascular Endothelial Cells with Listeria monocytogenes (Lm)

1. Advance preparation
   NOTE: Prepare the following solutions in advance.
   1. Streptomycin, chloramphenicol, and gentamicin stocks
      1. Prepare 50 mg/mL of streptomycin stock solutions by weighing 0.5 g of streptomycin sulfate and dissolving it completely into 10 mL of ultrapure water.
      2. Prepare 7.5 mg/mL of chloramphenicol stock solutions by weighing 75 mg of chloramphenicol and dissolving it completely into 10 mL of 100% ethanol.
      3. Prepare 20 mg/mL of gentamicin stock solutions by weighing 0.2 g of gentamicin sulfate and dissolving it completely into 10 mL of ultrapure water.
      4. Filter-sterilize all stock solutions with a 0.2-µm syringe filter and store them at -20 °C for long-term use (1 – 2 months).
   2. Brain heart infusion (BHI) media and agar plates
      1. Locate two 1-L flasks and add a magnetic stir bar inside each flask.
      2. For the BHI media, add 37 g of BHI powder in one flask and add ultrapure water up to 1 L. Mix the solution vigorously by placing the flask on a magnetic stir plate until the powder is dissolved.
      3. For the BHI agar plates, add 37 g of BHI powder and 15 g of granulated agar (see Table of Materials) in the second flask and add ultrapure water up to 1 L. Mix the solution vigorously by placing the flask on a magnetic stir plate until the powder is dissolved.
      4. Screw the lids of the flasks not too tightly and autoclave the solutions using the liquid setting or according to the autoclave's specifications.
      5. Remove the solution from the autoclave, then cool down the agar-BHI solution to 55 °C. If the BHI media in the 1 L flask is kept sterile, it can be used for up to a month.
      6. To prepare the bacteria agar-BHI plates, first add antibiotics, if appropriate, to the flask containing BHI and agar (depending on the bacterial strains to be grown). Briefly put the flask on a magnetic stir plate to allow quick mixing.         
         NOTE: The antibiotics used here are specific to the Lm strains used, but any necessary antibiotics can be used in BHI-agar plates. We added streptomycin to a concentration of 200 µg/mL and chloramphenicol to a concentration of 7.5 µg/mL because 10403S Lm strains are resistant to streptomycin. These strains have been conjugated with a plasmid that contains the chloramphenicol acetyltransferase open reading frame, hence the resistance to chloramphenicol.
      7. Pour the mixture into 10-cm polystyrene bacteria culture plates (approximately 20 mL per plate). To get rid of bubbles, flame the upper surface of the plates briefly. Cool plates overnight at room temperature.
      8. The next day, seal the dry plates and store them at 4 °C.
2. Infection of human microvascular endothelial cells with L. monocytogenes
   1. Three days before the infection, streak out the Lm strain to be used from a glycerol stock (stored at -80 °C) onto a BHI-agar plate that contains 7.5 µg/mL of chloramphenicol and 200 µg/mL of streptomycin, if appropriate.
      NOTE: The strain to be streaked out can be a wild-type or mutant, constitutively expressing fluorescence (for immunostaining JAT1045 was used) or expressing fluorescence under the ActA promoter (for flow cytometry or traction force microscopy JAT983 or JAT985 were used).
   2. Incubate plates at 37 °C until discrete colonies are formed (1 – 2 days).
   3. The day before the infection, grow the desired strain overnight, shaking it at 150 rpm at 30 °C in BHI media with 7.5 µg/mL of chloramphenicol (if appropriate).
      1. Place 5 mL of BHI media in a 15-mL conical centrifuge tube, add 7.5 µg/mL of chloramphenicol (if appropriate), and then inoculate a single colony from the agar plate using a sterile 10 µL tip.
   4. The next day, just prior to infection, measure the optical density of the bacteria solution at 600 nm (OD600) by diluting the sample 1:5; use a cuvette containing BHI alone to serve as a blank.
   5. Dilute the overnight culture to an OD600 of 0.1 and incubate it, shaking for 2 h at 30 °C, in BHI media with 7.5 µg/mL of chloramphenicol (if appropriate) to allow the bacteria to reach log-phase growth.
   6. Measure the OD600 of the bacterial solution, which is expected to be around 0.2-0.3. If the OD600 is higher, dilute it to 0.2 – 0.3 with BHI alone.
   7. Take 1 mL of bacterial solution into a microcentrifuge tube. Spin it down for 4 min at 2,000 x g using centrifugation at room temperature. Remove the supernatant and resuspend the bacterial pellet in 1 mL of tissue culture-grade PBS, in the tissue culture hood. Wash the bacteria twice more by spinning them down for 4 min at 2,000 x g at room temperature. Remove the supernatant and resuspend the bacteria in 1 mL of PBS.
   8. Prepare the infection mix by mixing 10 or 50 µL of the bacteria resuspended in PBS with 1 mL of MCDB-131 full media for a multiplicity of infection (MOI; i.e., the number of bacteria per host cell) of approximately 50 bacteria per host cell or 10 bacteria per host cell.
   9. Remove the media from the wells of the 24-well plates, careful not to disrupt the hydrogels or the cells. Wash the cells 1x with 1 mL of MCDB-131 full media and then add 1 mL of the bacteria to each well.
   10. Keep some infection mix (at least 100 µL) for the determination of MOI (see step 3.3).
   11. Cover the plate with its lid and wrap the plates with polyethylene food wrap to avoid leakage. Place the plates in the centrifuge and spin the samples for 10 min at 200 x g to synchronize the invasion. Move the plates into the tissue culture incubator and incubate them for 30 min at 37 °C.
   12. Wash the samples 4x with MCDB-131 full media and move them into the tissue culture incubator. After an additional 30 min, replace the media with media supplemented with 20 µg/mL of gentamicin.
3. Determination of the multiplicity of infection (MOI)
   1. During the initial 30-min incubation of the host cells with the bacteria, prepare 10-fold serial dilutions of the infection mix to determine the MOI. Make 10-fold dilutions by mixing 100 µL of the infection mix with 900 µL of 1x PBS (10^-1 dilution). Then, mix 100 µL of the 10^-1 dilution with 900 µL of PBS (10^-2 dilution).
   2. Continue until a 10^-5 dilution is obtained.
      NOTE: All solutions need to be mixed well before diluting them, and fresh clean pipette tips should be used at each step.
   3. Once the dilutions are made, place 100 µL of the 10^-2 – 10^-5 dilutions on the center of the BHI/agar/chloramphenicol/streptomycin plates (if appropriate).
   4. Make a spreader from a glass pipette by using fire to bend the pipette to create a hook. Dip the pipette in 100% ethanol for sterilization and then burn off the ethanol with a flame.
   5. Once the spreader has cooled, spread the bacterial dilutions homogeneously on the plates starting from the 10^-5 dilution and moving to the more concentrated mixes. Incubate the plates upside down at 37 °C for 2 days.
   6. Depending on the colony density, determine the number of colonies of either the 10^-3 and 10^-4 or the 10^-4 and 10^-5 dilution plates. Calculate the MOI as follows:     
      number of colonies × dilution factor ÷ volume initial added = number of colony forming unit (CFU) per µL     
      number of CFU per µL × volume of bacteria mix per well = number of CFU per well      
      number of CFU per well × volume of cells per well = number of bacteria per cell
      NOTE: CFU stands for colony-forming units.
   7. Average the MOIs from two plates to obtain the final MOIs.

4. Quantitative Time-lapse Microscopy to Assess Extracellular-matrix-stiffness

Prepare the PA hydrogels on a 24-well plate (see step 1), seed HMEC-1 cells (see step 2), and grow overnight JAT983 cultures as described above (see step 3).

1. The day of infection, prepare the infection mix by mixing 10 µL of bacterial pellet per mL of MCDB-131 full media. Carefully remove the media from the wells of the 24-well plates, and add 1.9 mL of the MCDB-131 full media and 0.1 mL of the bacterial mix to each well.  
   NOTE: The lower MOI used in this assay is necessary to analyze dissemination events that start from a single bacterium invading a host cell.
2. Cover the plate with its lid and seal it with a plastic wrap to avoid leakage. Place the plates in the centrifuge and spin the samples for 10 min at 200 x g to synchronize the invasion.
3. Move the plates into the tissue culture incubator and incubate them for 5 min.
4. Wash the samples 4x with media and move them to the tissue culture incubator. After an additional 5 – 7 min, replace the media with media supplemented with 20 µg/mL of gentamicin.
5. Incubate the plate in the incubator for an extra 5 h to allow the actA promoter to turn on and drive the expression of the mTagRFP open reading frame.
6. Prepare a live-microscopy medium by supplementing Leibovitz's L-15 with 10% fetal bovine serum, 10 ng/mL of epidermal growth factor, and 1 µg/mL hydrocortisone.
   NOTE: The L-15 already contains 2 mM L-glutamine, so it is not necessary to add extra as in the case of MCDB-131 media.
7. Mix 1 μL of 1 mg/mL Hoechst dye with 1 mL of L-15 full media and add it to each well to stain the cells' nuclei. Place the cells in the tissue culture incubator for 10 min and then replace in each well the L-15 full media with 1 mL of L-15 full media supplemented with 20 µg/mL of gentamicin. Cover the plate with its lid and place it in a microscope's environmental chamber (see Table of Materials) equilibrated to 37 ˚C.      
   NOTE: For imaging, an inverted epifluorescence microscope was used with a CCD camera (see Table of Materials) and a 20X air plan fluorite air objective with 0.75 NA. The power was set to 50% and the exposure time to 50 ms. The microscope filters used for mCherry are 470/525 nm for excitation and emission respectively.
8. Image multiple positions every 5 min using an autofocus feature to monitor how Lm spread through HMEC-1 monolayers seeded on varying stiffness hydrogels.
   NOTE: L-15 is buffered with HEPES, so it is not necessary to use CO₂ during the imaging.

Table 1. Composition of polyacrylamide (PA) hydrogels of varying stiffness. In this table, the percentage of stock 40% acrylamide solution and the percentage of stock 2% bis-acrylamide solution to achieve a given stiffness (Young's modulus, E) are indicated in different columns.