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

Take a multi-well plate with adherent human endothelial cell monolayers on collagen-coated polyacrylamide hydrogels of varying stiffness.

Cells on stiffer hydrogels undergo significant cytoskeletal reorganization, increasing intracellular tension, compared to softer hydrogels.

Wash cells with media and add engineered Listeria monocytogenes — expressing a fluorescent protein under the actin assembly-inducing protein (ActA) promoter.

Centrifuge to facilitate cell-bacteria contact. Incubate to mediate bacterial endocytosis.

Wash with media. Treat with gentamicin to eliminate non-internalized bacteria. Incubate.

Internalized bacteria release pore-forming toxins, disrupting phagosomal membranes. Cytosolic bacteria upregulate ActA and fluorescent protein expression, labeling intracellular bacteria.

ActA proteins induce actin polymerization, forming an actin comet tail propelling the bacteria forward, causing internalization by neighboring cells, and promoting dissemination.

Post-incubation, use dye-containing media to stain cell nuclei. Replace media with media containing gentamicin.

Perform time-lapse microscopy.

Softer hydrogels promote rapid bacteria intercellular spread compared to stiffer hydrogels with increased intracellular tension, suggesting matrix stiffness-dependent bacterial dissemination.

After preparing an overnight culture of L. monocytogenes according to the text protocol, transfer 1 milliliter of the culture into a microcentrifuge tube, and spin it down at 2,000 times g and room temperature for 4 minutes. After using tissue culture-grade PBS to wash the pellet twice, use 1 milliliter of PBS to resuspend the pellet.

Prepare the infection mix by combining 10 or 50 microliters of the bacterial suspension with 1 milliliter of MCDB-131 full medium for a multiplicity of infection, or MOI, of approximately 50 bacteria per host cell or 10 bacteria per host cell. Remove the medium from the wells of the 24-well plates, taking care not to disrupt the hydrogels or the cells.

Use 1 milliliter of MCDB-131 full medium to wash the cells once, then add 1 milliliter of the bacteria to each well. Place the lid on the plates, and wrap them with polyethylene food wrap to avoid leakage. Centrifuge the plates at 2,000 x g for 10 minutes to synchronize the invasion. Then, incubate the cultures at 37 degrees Celsius for 30 minutes. With MCDB-131 full medium, wash the samples 4 times and return them to the tissue culture incubator. After an additional 30 minutes, replace the medium with MCDB-131 full medium supplemented with 20 micrograms per milliliter of gentamicin.

After seeding HMEC-1 cells on PA hydrogels and treating with gentamicin according to the text protocol, incubate the plate for five hours to allow the ActA promoter to turn on and drive the expression of the mTagRFP open reading frame.

Four hours post-infection, mix 1 microliter of 1mg/ml Hoechst dye with 1 milliliter of L15 full medium and add it to each well to stain the nuclei. After incubating the cells for 10 minutes, replace the medium with 1 milliliter of L15 full medium supplemented with 20 micrograms per milliliter of gentamicin.

Image multiple positions every 5 minutes using an autofocus feature to monitor how LM bacteria spread through HMEC-1 monolayers seeded on varying stiffness hydrogels.