An Assay to Study the Impact of Extracellular Matrix Stiffness on Bacterial Infection

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 times 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.

To carry out flow cytometry eight hours post-infection, remove the medium from the wells of the 24-well plate, and use tissue culture PBS to wash the wells once. Following the removal of the PBS, add 200 microliters of trypsin-EDTA-collagenase mix to each well. Place the dish in the tissue culture incubator for 10 minutes to allow full detachment of the cells.

After gently pipetting each well eight times, add 200 microliters of full medium to neutralize the trypsin. Transfer the 400 microliters of cell solution from each well into a 5-milliliter polystyrene tube with a 35-micrometer cell strainer cap. Analyze the samples by flow cytometry.