The overall goal of this methodology is to enable the characterization of the effect of extracellular matrix stiffness on bacterial infection of adherent cells in a highly quantitative manner. This method can help answer key questions in the emerging field of host-pathogen biomechanics, such as what is the role of mechanical forces in modulating bacterial infection susceptibility of host cells. This technique helps create high-resolution time lapse video sequences while simultaneously screening multiple conditions and automating certain procedures.

To carry out glass activation of 24-well dishes, add 500 microliters of tumalo sodium hydroxide per 13-millimeter diameter well and incubate the plates at room temperature for one hour. Discard the sodium hydroxide and use ultrapure water to rinse the wells once. Then add 500 microliters of two-percent triethoxysilane in 95%ethanol to each well and incubate them for five minutes.

With water, rinse the wells once. Then add 500 microliters of 0.5%glutaraldehyde to each well and incubate the plates for 30 minutes. After rinsing once with water, dry the plates at 60 degrees Celsius with the lid off.

To manufacture hydrogels of tunable stiffness, prepare aqueous solutions that contain three to 10 percent of a 40%stock acrylamide solution and 06 to 6 percent of a two percent bis-acrylamide solution, depending on the desired stiffness of the hydrogel. After this, add the water. For each stiffness, Solution One is bead-free, whereas Solution Two contains 03%0.1-micrometer fluorescent microbeads.

Degas Solutions One and Two by vacuum for 15 minutes to eliminate oxygen that will inhibit polymerization. Then, while acting quickly, add 0.43%TEMED and 0.6%of the 10-gram-per-milliliter stock APS solution to Solution One. Add 3.6 microliters of the solution to the center of each well of the 24-well dish.

Immediately use 12-millimeter circular cover slips to cover the wells and let the solution sit for 20 minutes so that it fully polymerizes. Gently tap a syringe needle on a hard surface to create a small hook at its tip to facilitate the removal of the cover slips, then use the needle to lift the cover slips. Next, add 0.43%TEMED and 0.6%of the 10-gram-per-milliliter stock APS solution to Solution Two.

Then deposit 2.4 microliters of the mixture on top of the 12-millimeter circular cover slips. Place the circular cover slips with a drop of Solution Two on top of the first polyacrylamide layer and use forceps to gently press downwards to ensure the thickness of the second layer is minimal. Then let Solution Two polymerize for 20 minutes.

Add 500 microliters of 50 millimolar HEPES pH 7.5 to each of the wells and use the syringe needle and forceps to remove the glass cover slips. To sterilize the hydrogels, place them in a tissue culture hood and expose them to UV for one hour. Now, prepare a mixture of 0.5%weight-per-volume of Sulfo-SANPAH and one percent DMSO and 50 millimolar HEPES pH 7.5.

Add 200 microliters of the solution to the upper surface of the hydrogels. Then working quickly, expose them 302 nanometers UV for 10 minutes to activate them. Use one milliliter of 50 millimolar HEPES pH 7.5 to wash the hydrogels twice, repeating if necessary to remove any excess cross-linker.

Protein-coat the hydrogels with 200 microliters of 0.25 milligrams-per-milliliter rat tail collagen I and 50 millimolar HEPES. Incubate the hydrogels with the collagen at room temperature overnight. Before seeding the cells of interest on the hydrogels, add one milliliter of medium and equilibrate them at 37 degrees Celsius for one hour.

To seed human microvascular endothelial cells, after culturing and preparing a cell suspension according to the text protocol, remove the medium from the hydrogels, then add one milliliter of cell suspension to each well. After preparing an overnight culture of L.monocytogenes according to the text protocol, transfer one milliliter of the culture into a microcentrifuge tube and spin it down at 2, 000 times g at room temperature for four minutes. After using tissue-culture grade PBS to wash the pellet twice, use one milliliter of PBS to re-suspend the pellet.

Prepare the infection mix by combining 10 or 50 microliters of the bacterial suspension with one 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 one milliliter of MCDB 131 full medium to wash the cells once, then add one 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 four 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 five-milliliter polystyrene tube with a 35-micrometer cell strainer cap.

Analyze the samples by flow cytometry. 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 one microliter of one milligram per milliliter Hoechst dye with one milliliter of L-15 full medium and add it to each well to stain the nuclei.

After incubating the cells for 10 minutes, replace the medium with one milliliter of L-15 full medium supplemented with 20 micrograms per milliliter of gentamicin. Image multiple positions every five minutes using an auto-focus feature to monitor how LM bacteria spread through HMEC-1 monolayers seeded on varying stiffness hydrogels. As reported in this graph, AFM measurements were performed to confirm the exact stiffness of the Pa hydrogels prepared using the protocol in this video.

Here, HMEC-1 cells on matricies of different stiffness were infected with an LM strain that expresses a fluorescent marker after internalization that only allows the detection of intracellular bacteria. Cells were gated using the forward-versus-side scatter plot and a second gating step excluded cells that exhibited autofluorescence. Flow cytometry analysis revealed that LM infection was approximately two-fold greater on stiff 70 kilopascal versus 0.6 kilopascal hydrogels.

To test whether increased LM adhesion onto HMEC-1 increased LM invasion into HMEC-1, or both, were responsible for the increased susceptibility to infection shortly after infection with LM constitutively expressing GFP, the HMEC-1 cells were fixed and adhered bacteria were stained with antibodies. As shown here, there were significantly more bacteria adhering to HMEC-1 when the host cells reside on stiff as compared to soft gels. Consistent with the flow cytometry data, there are significantly more bacteria internalized by HMEC-1 when the host cells reside on stiff as compared to soft gels.

Once mastered, this assay can be done in approximately three days, as there are long incubation steps required in between. While attempting this procedure, it is important to remember to be as sterile as possible to ensure that hydrogels and host cells are contamination-free. Following this procedure, other methods such as atomic force microscopy can also be incorporated to answer questions such as how does the stiffness of host cells change upon infection and whether this effect depends on matrix stiffness.

After its development, this technique paved the way for researchers in the field of mechanobiology to explore the role of host cell pathogen biomechanical interactions by using different host cells, such as the epithelial cells, and different bacterial pathogens, such as Rickettsia parkeri. After watching this video, you should have a good understanding on how to manufacture hydrogels of tunable stiffness on multi-welled plates and how to perform the infection assay. Don't forget that working with pathogenic bacteria can be hazardous.

Therefore, precautions, such as inserting barriers between the site of entry and the pathogen, as well as prevent the generation of aerosols should always be taken while performing this procedure.

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