Name: a multi-well format polyacrylamide-based assay for studying the effect of extracellular matrix stiffness on the bacterial infection of adherent cells
Uploaded: 2018-07-05T13:00:00
Duration: 11 min 51 s
Description: Watch this Scientific Journal Video about A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells at JoVE.

Our thanks to M. Footer, R. Lamason, M. Rengaranjan, and members of the Theriot Lab for their discussions and experimental support. This work was supported by NIH R01AI036929 (J.A.T.), HHMI (J.A.T.), the HHMI Gilliam Fellowship for Advanced Study (F.E.O.), the Stanford Graduate Fellowship (F.E.O.),&#160;the American Heart Association (E.E.B.), NIH 1R01HL128630 (R.S.), and NIH 1R01HL130840 (R.S.). Flow cytometry was performed at the Stanford Shared FACS Facility.

The authors have nothing to disclose.

Cells can sense a variety of physical environmental cues, which can affect not only the cells&#39; morphology but also their gene expression and protein activity, thus affecting critical cell functions and behaviors45,46. The stiffness of the ECM of cells is increasingly appreciated as an important modulator of cellular motility, differentiation, proliferation, and ultimately cell fate47,48,49. Although there have been many recent advances in understanding the complex biomechanical interaction between cells and their ECM, little is known about how environmental stiffness affects the susceptibility of cells to bacterial infection. To facilitate such studies, we developed this novel multi-well assay based on the well-established fabrication of polyacrylamide hydrogels of tunable stiffness which is compatible with infection assays50. Traditionally, a bacterial infection of cells in tissue culture has been studied on glass or polystyrene surfaces that are approximately 1 - 3 orders of magnitude stiffer than the natural ECM of most adherent cells8,51. The assay described here opens new highways by enabling the study of bacteria-host interactions in a physiologically relevant environmental stiffness regime.
For proof of concept of the presented assay, HMEC-1 cells were chosen as model adherent host cells and Lm as a model bacterial pathogen. However, the assay can be extended for further studies if appropriately modified. Such studies can involve infection of different mammalian adherent host cell types by additional pathogens, including bacteria and viruses. For this particular assay, gels were protein-coated with collagen I, but depending on the host cell type, it is possible to use a different ECM protein-coating, such as laminin or fibronectin, to facilitate the attachment of the host cells of interest on the hydrogel52. An additional consideration that depends on the host cell type is the hydrogel stiffness range to be studied. The range of stiffness should depend on what is physiologically relevant for the specific host cell type and how well that host attaches forming a monolayer at a given stiffness hydrogel. Similarly, depending on the model pathogen desired to be examined, slight modifications might need to be implemented on the infection assay described herein.
The innovation of the assay as compared to previous methods for manufacturing polyacrylamide hydrogels24,50,53 lies in certain unique features integrated into the proposed infection assay. First, the hydrogels are built on multi-well glass bottom plates, which enables the screening of multiple conditions simultaneously as well as the automation of certain procedures. Monitoring multiple conditions at the same time or examining multiple replicates is crucial since the outcome of such an approach can be influenced by factors such as the host cell passage and the precise number of bacteria added to infect the hosts, which often change between independent experiments. An additional unique feature of this assay is that the hydrogels have a height of approximately 40 &#181;m, which is thin enough to image using conventional microscopy. We showed that we can successfully perform both live cell microscopy and cell fixation, and immunostaining followed by imaging, without introducing high background fluorescence. Lastly, the hydrogels are made of two layers, with the upper one having embedded fluorescent microbeads, confined in a single focal plane. This attribute ensures that there will be no out-of-focus light interfering during imaging. The presence of the beads enables both the examination of the surface topography of the gels to ensure that they are uniform and improves the performance of TFM and MSM24,32,41. With TFM and MSM, it is possible to calculate the cell-ECM and cell-cell forces respectively of cells residing on varying stiffness matrices. Using this novel assay, it is possible to make such measurements of physical forces by comparing both the contribution of environmental stiffness and of infection simultaneously. Following such an approach, the effect infection has on host cell mechanics throughout its course can be determined. Additionally, the evolution of the intracellular stresses of cells can be calculated using MSM and can be used as a measure of the barrier integrity of the monolayer. Finally, given the multi-well nature of the assay, it is possible to simultaneously introduce pharmacological and genetic perturbations with a bacterial infection, to investigate in more depth the complex interplay between host cell mechanics and infection.
One inherent limitation of the technique lies in the effect substrate stiffness might have on the proliferation rate of cells. Typically, in infection assays, we need to ensure that the host cell density under different conditions is the same. That is because cell density by itself can have an effect on the susceptibility of hosts to infection. The HMEC-1 cells that were used as host cells do not show a significant difference in their number when seeded for 24 h on the hydrogels. However, different cell types might exhibit differential proliferation, depending on the hydrogel stiffness, that can bias infection studies. Similarly, infection bias can arise when cells do not form monolayers or do not attach well on the hydrogels, as occurs when certain cell types are seeded on very soft matrices (e.g., human umbilical cord endothelial cells or Madin-Darby canine kidney epithelial cells seeded on 0.6-kPa hydrogels54,55). As far as pathogens are concerned, certain bacteria (e.g., Borrelia burdgorferi) can attach onto host cells and invade them but can also transmigrate through them56. We have not yet tested if this assay would work for pathogen transmigration studies, but it seems possible since previous studies on neutrophils transmigrating endothelial cells seeded on PA hydrogels have been documented to work successfully57. There have been a lot of studies conducted showing how to manufacture polyacrylamide hydrogels of a given Young&#39;s modulus by mixing the appropriate concentrations of acrylamide and bis-acrylamide24,25,26,27. However, especially when one desires to perform TFM experiments on cells residing on hydrogels of varying stiffness, it is critical to confirm the expected stiffness of the hydrogels either through AFM or other indentation techniques58. Slight deviations from the expected value can arise due to a different solvent used or an aged acrylamide stock solution, or by the ways AFM measurements are performed (e.g., the shape of the AFM tip). Finally, the approach presented herein is based on seeding host cells on 2D matrices, which can differ from a more realistic and physiologically relevant 3D scenario. However, manufacturing 3D gels with tunable stiffness, seeding them with host cells and then infecting them with pathogens, still encompasses certain technical difficulties. Nevertheless, we anticipate that in the near future we will be able to extend the current assay for studying infection in a 3D setting.
To sum up, the described protocol together with the preliminary results provide evidence that this novel assay can become an extremely useful tool for studying infection of adherent host cells with pathogenic bacteria in a quantitative fashion and in a much more physiologically relevant environment than previously examined. The power of fabricating polyacrylamide hydrogels in the proposed setup lies in that the assay is compatible with the performance of multiple techniques such as flow cytometry, immunostaining followed by light microscopy, and traction force microscopy. The assay can be used for studies involving the infection of different adherent host cell types by pathogens, which we anticipate will have a significant impact in both unraveling the strategies by which pathogens infect hosts and in facilitating the development of therapeutic interventions against infections.

Cells in most animal tissues are typically adherent, attaching both to neighboring cells and to their extracellular matrix (ECM). The anchorage of cells to their ECM is critical for many cellular processes ranging from cell motility to cell proliferation and survival1,2. Cellular anchorage to the ECM depends on both the ECM composition and stiffness. Cells respond to changes in the latter by dynamically re-arranging their cytoskeleton, cell-ECM and cell-cell adhesions, which in turn critically alter cell biomechanics and functions3,4,5,6. ECM stiffness can vary in space (i.e., anatomic location), in time (i.e., aging), and in pathophysiological processes (e.g., arteriosclerosis, cancer, infections, etc.). For instance, it is widely accepted that endothelial cells residing on stiffer-as compared to softer-matrices exert increased forces to their ECM and to each other and exhibit increased motility and proliferation7,8. Likewise, fibroblasts residing on stiffer matrices impart high contractile forces to their ECM and show increased proliferation, motility, and ECM production9,10,11. Although cell mechanics and response to ECM stiffness have been studied extensively for various cell types, the relationship between adherent host cells, the stiffness of their ECM, and bacterial infections is still largely unknown.
To study the role of ECM stiffness in bacteria-host cell interactions, L. monocytogenes (Lm) was chosen as the model pathogen. Lm is a ubiquitous food-borne bacterium that can cause systemic infection in a wide variety of mammalian hosts. This facultative intracellular pathogen can move from the intestinal epithelium to distant organs by traversing different types of vascular endothelia. If it breaches the blood-brain barrier, Lm can cause meningitis, and when it crosses the placenta, it can cause spontaneous abortion12,13. Lm can infect different host cell types and can do so by using distinct pathogenic strategies. Lm infection has been studied mostly in the context of epithelial cells, while much less is known about how Lm can infect and bypass endothelial cells lining the lumen of blood vessels14,15,16. Moreover, it is still largely unknown how the stiffness of the ECM where endothelial cells reside modulates Lm&#39;s ability to invade these host cells and to then spread. Lm and several additional bacterial species (i.e., Rickettsia parkeri) take advantage of the actin cytoskeleton of the host cells they infect to both invade into their cytoplasm and facilitate cell-to-cell dissemination17,18,19. They achieve that through the expression of proteins that enable them to interfere with host actin polymerization pathways and to produce actin comet tails that facilitate their forward propulsion16,20. As a result of infection, the host cell&#39;s actin cytoskeleton needs to dynamically rearrange in a manner that is still not fully characterized, potentially affecting the biomechanics of the host cells including the physical forces they exert on their ECM and on each other. To examine these processes, human microvascular endothelial cells (HMEC-1) were chosen as model host cells for three reasons: 1. endothelial cells are known to be highly mechanosensitive as they are constantly exposed to varying physical cues21; 2. the strategies Lm employs to infect endothelial cells are still largely unknown22; and 3. HMEC-1 are an immortalized cell line and can, therefore, be easily cultured and subjected to genetic manipulation.
Bacterial infection of host cells has mostly been studied in vitro by seeding cells on glass or polystyrene substrates that are significantly stiffer than the physiological ECM of most cells12,14,23. To examine the infection of cells seeded on a matrix whose stiffness is physiologically relevant and to elucidate the role of ECM stiffness on the infection of cells by bacterial pathogens, we followed an innovative approach based on fabricating thin microbead-embedded polyacrylamide hydrogels of tunable stiffness on multi-well plates. The novelty of the proposed approach lies in that it allows monitoring multiple conditions simultaneously due to its multi-well format and in that it is compatible with multiple techniques due to the particular way the substrates are built. HMEC-1 cells were seeded on these protein-coated hydrogels and then infected with different Lm strains that either become fluorescent upon internalization or are constitutively fluorescent. The role of ECM stiffness on infection susceptibility of host HMEC-1 cells was evaluated by flow cytometry. In addition, immunostaining and fluorescence microscopy were used to differentiate between adhering and internalized bacteria. Finally, Traction Force Microscopy (TFM) was successfully performed to characterize the effect of Lm infection on the traction stresses that host endothelial cells exert on their matrices during infection. The presented assay can be easily modified to enable further studies on the effect of ECM stiffness on infection susceptibility of adherent cells using different cell lines or pathogens.

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			<td height="20" style="height:20px;width:355px;">Reagents</td>
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			<td height="20" style="height:20px;width:355px;">Sodium hydroxide pellets</td>
			<td>Fisher</td>
			<td style="width:151px;">S318-500</td>
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			<td height="20" style="height:20px;width:355px;">Bis-acrylamide solution (2%w/v)</td>
			<td>Fisher Scientific</td>
			<td style="width:151px;">BP1404-250</td>
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			<td height="20" style="height:20px;">Fluorospheres carboxylate-modified microspheres, 0.1 &#956;m, yellow-green fluorescent (505/515)</td>
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			<td>BP17925</td>
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			<td style="width:149px;">Sigma</td>
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			<td height="20" style="height:20px;width:355px;">HEPES, Free acid</td>
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			<td style="width:151px;">21083027</td>
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			<td>Life technologies</td>
			<td style="width:151px;">10372019</td>
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			<td height="20" style="height:20px;">Foundation Fetal Bovine Serum, Lot: A37C48A</td>
			<td>Gemini Bio-Prod</td>
			<td>900108 500ml</td>
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			<td style="width:149px;">Sigma</td>
			<td style="width:151px;">H0888</td>
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			<td height="20" style="height:20px;">L-Glutamine 200mM</td>
			<td>Fisher</td>
			<td>SH3003401</td>
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			<td height="20" style="height:20px;">DPBS 1X</td>
			<td>Fisher</td>
			<td>SH30028FS</td>
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			<td height="20" style="height:20px;">Gentamicin sulfate</td>
			<td>MP biomedicals</td>
			<td>194530</td>
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			<td height="20" style="height:20px;">Cloramphenicol</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:151px;">C0378-5G</td>
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			<td style="width:149px;">BD</td>
			<td style="width:151px;">214530</td>
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			<td style="width:149px;">BD</td>
			<td style="width:151px;">211059</td>
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			<td height="20" style="height:20px;">Hoechst 33342, trihydrochloride, trihydrate - 10 mg/ul solution in water</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td></td>
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			<td height="20" style="height:20px;">Formaldehyde 16% EM grade</td>
			<td>Electron microscopy</td>
			<td>15710-S</td>
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			<td style="width:149px;">Abcam</td>
			<td style="width:151px;">ab35132</td>
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			<td>Thermofischer</td>
			<td style="width:151px;">A-11035</td>
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			<td height="20" style="height:20px;">0.25% trypsin-EDTA , phenol red</td>
			<td>Thermofischer</td>
			<td style="width:151px;">25200056</td>
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			<td height="20" style="height:20px;">COLLAGENASE FROM CLOSTRIDIUM HISTOLYTIC</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:151px;">C8051</td>
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			<td height="20" style="height:20px;">Streptomycin sulfate</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:151px;">3810-74-0</td>
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			<td style="width:149px;">Thermofischer</td>
			<td>28364</td>
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			<td height="20" style="height:20px;width:355px;">MES powder</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:151px;">M3885</td>
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			<td height="20" style="height:20px;">KCl</td>
			<td style="width:149px;">J.T. Baker</td>
			<td style="width:151px;">3040-05</td>
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			<td style="width:149px;">J.T. Baker</td>
			<td style="width:151px;">2444-1</td>
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			<td style="width:149px;">Acros</td>
			<td style="width:151px;">40991</td>
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			<td style="width:149px;"></td>
			<td style="width:151px;"></td>
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			<td height="20" style="height:20px;width:355px;">12 mm circular glass coverslips</td>
			<td style="width:149px;">Fisherbrand</td>
			<td style="width:151px;">12-545-81</td>
			<td>No. 1.5 Coverslip | 10 mm Glass Diameter | Uncoated</td>
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			<td height="20" style="height:20px;">Glass bottom 24 well plates</td>
			<td style="width:149px;">Mattek</td>
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			<td height="20" style="height:20px;">5 ml polystyrene tubes with a 35 &#956;m cell strainer cap&#160;</td>
			<td style="width:149px;">Falcon</td>
			<td style="width:151px;">352235</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:355px;">T-25 flasks</td>
			<td style="width:149px;">Falcon</td>
			<td style="width:151px;">353118</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:355px;">50 ml conical tubes</td>
			<td style="width:149px;">Falcon</td>
			<td style="width:151px;">352070</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:355px;">15 ml conicals tubes</td>
			<td style="width:149px;">Falcon</td>
			<td style="width:151px;">352196</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Disposable Serological Pipettes (1 ml, 2 ml, 5 ml, 10 ml, 25 ml)</td>
			<td style="width:149px;">Falcon</td>
			<td style="width:151px;">357551</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Pasteur Glass Pipettes</td>
			<td style="width:149px;">VWR</td>
			<td>14672-380</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Pipette Tips (1-200 &#956;l, 101-1000 &#956;l)</td>
			<td style="width:149px;">Denville</td>
			<td style="width:151px;">P1122, P1126</td>
			<td></td>
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			<td height="20" style="height:20px;width:355px;">Powder Free Examination Gloves</td>
			<td style="width:149px;">Microflex</td>
			<td style="width:151px;">XC-310</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Cuvettes bacteria</td>
			<td style="width:149px;">Sarstedt</td>
			<td style="width:151px;">67.746</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Razors</td>
			<td style="width:149px;">VWR</td>
			<td>55411-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Syringe needle</td>
			<td style="width:149px;">BD</td>
			<td style="width:151px;">305167</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:355px;">0.2um sterilizng bottles</td>
			<td style="width:149px;">Thermo Scientific</td>
			<td>566-0020</td>
			<td></td>
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			<td height="20" style="height:20px;width:355px;">20 ml syringes</td>
			<td style="width:149px;">BD</td>
			<td style="width:151px;">302830</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:355px;">0.2um filters</td>
			<td style="width:149px;">Thermo Scientific</td>
			<td style="width:151px;">723-2520</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:355px;">wooden sticks</td>
			<td style="width:149px;">Grainger</td>
			<td>42181501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Saran wrap</td>
			<td style="width:149px;">Santa Cruz Biotechnologies</td>
			<td style="width:151px;">sc-3687</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Plates bacteria</td>
			<td style="width:149px;">Falcon</td>
			<td style="width:151px;">351029</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Large/non-disposable lab equipment</td>
			<td style="width:149px;"></td>
			<td style="width:151px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Tissue Culture Hood</td>
			<td style="width:149px;">Baker</td>
			<td style="width:151px;">SG504</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Hemacytometer</td>
			<td style="width:149px;">Sigma</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Bacteria incubator</td>
			<td style="width:149px;">Thermo Scientific</td>
			<td>IGS180</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Tissue culture Incubator</td>
			<td style="width:149px;">NuAire</td>
			<td style="width:151px;">NU-8700</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:355px;">Vacuum chamber/degasser</td>
			<td style="width:149px;">Belart</td>
			<td style="width:151px;">42025</td>
			<td>37&#730;C and 5% CO2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Inverted Nikon Diaphot 200 epifluorescence microscope</td>
			<td style="width:149px;">Nikon</td>
			<td>NIKON-DIAPHOT-200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cage Incubator</td>
			<td style="width:149px;">Haison</td>
			<td style="width:151px;">Custom</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scanford FACScan analyzer&#160;</td>
			<td>Stanford and Cytek upgraded FACScan</td>
			<td style="width:151px;">Custom</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Pipette Aid</td>
			<td style="width:149px;">Drummond</td>
			<td>4-000-110</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Pipettors (10 &#956;l, 200 &#956;l, 1000ul)</td>
			<td style="width:149px;">Gilson</td>
			<td colspan="2">F144802, F123601, F123602</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">pH meter</td>
			<td style="width:149px;">Mettler Toledo</td>
			<td>30019028</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">forceps</td>
			<td style="width:149px;">FST</td>
			<td>11000-12</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">1 L flask</td>
			<td style="width:149px;">Fisherbrand</td>
			<td>FB5011000&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Autoclave machine</td>
			<td style="width:149px;">Amsco</td>
			<td style="width:151px;">3021</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Stir magnet plate</td>
			<td style="width:149px;">Bellco</td>
			<td>7760-06000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Magnet stirring bars</td>
			<td style="width:149px;">Bellco</td>
			<td>1975-00100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Spectrophotometer</td>
			<td style="width:149px;">Beckman</td>
			<td style="width:151px;">DU 640</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:355px;">Scanford FACScan analyzer</td>
			<td style="width:149px;">Cytek Biosciences</td>
			<td style="width:151px;">Custom Stanford and Cytek upgraded FACScan</td>
			<td></td>
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			<td height="20" style="height:20px;width:355px;">Software</td>
			<td style="width:149px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Microscope Software (&#956;Manager)</td>
			<td style="width:149px;">Open Imaging</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Matlab</td>
			<td style="width:149px;">Matlab Inc</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Flowjo</td>
			<td style="width:149px;">FlowJo, LLC</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:355px;">Automated image analysis software, CellC</td>
			<td style="width:149px;">https://sites.google.com/site/cellcsoftware/</td>
			<td style="width:151px;"></td>
			<td>The software is freely available. Eexecutable files and MATLAB source codes can be obtained at https://sites.google.com/site/cellcsoftware/</td>
		</tr>
	</tbody>
</table>

a multi-well format polyacrylamide-based assay for studying the effect of extracellular matrix stiffness on the bacterial infection of adherent cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

ECM stiffness-dependent susceptibility of HMEC-1 cells to Lm infection: 
PA hydrogels of varying stiffness, all surface-coated with collagen I, were built on multi-well glass bottom plates as described in step 1 of the protocol (see Figure 1). AFM measurements were performed to confirm the exact stiffness of the hydrogels, as described previously26,27 (see Figure 2). Past studies have shown that the local compliance of the basement membrane of endothelial cells can range from 1 kPa (e.g., brain tissue) to 70 kPa (e.g., aorta)36,37,38,39,40. Therefore, we chose to test the infection of HMEC-1 cells residing on matrices with the following stiffness: 0.6 kPa, 3 kPa, 20 kPa, and 70 kPa. For each hydrogel stiffness, six hydrogels were fabricated to assess the reproducibility of the results.
Flow cytometry was used to assess the ECM stiffness-dependent susceptibility of HMEC-1 cells to Lm infection (see Figure 3). HMEC-1 cells were infected with a Lm strain (JAT985) that expresses a fluorescent marker after internalization (actAp::mTagRFP), allowing the detection of intracellular bacteria only (see Figures 1L - 1N). JAT985 also lacks ActA, disabling the bacteria from spreading from cell to cell, since ActA is necessary for the formation of actin comet tails and subsequent bacterial dissemination. 7 - 8 h post-infection, the HMEC-1 cells&#39; infection was assessed using flow cytometry. To ensure the analysis of single cells, the bulk of the distribution of cell counts was gated using the forward versus side scatter plot, and then a second gating step was performed to exclude cells that exhibit autofluorescence (see Figures 3A - 3C). The preliminary results depicted in Figure 3 show that HMEC-1 infection with Lm is approximately two-fold greater for HMEC-1 cells residing on the stiff 70 kPa hydrogels as compared to cells residing on the soft 0.6 kPa hydrogels (see Figures 3B - 3D). The increased Lm infection susceptibility of HMEC-1 cells residing on stiff as compared to soft matrices could be due to: 1. increased Lm adhesion onto HMEC-1; 2. increased Lm invasion into HMEC-1; or 3. both of the above co-occurring. To test which hypothesis holds, HMEC-1 cells were seeded on soft (3 kPa) and stiff (70 kPa) hydrogels and infected with constitutively GFP expressing Lm (see Figures 4A - 4C). The samples were fixed shortly after infection and the external (adhering) bacteria were stained with antibodies. Using quantitative microscopy, we found that there are significantly more bacteria adhering to HMEC-1 when the host cells reside on stiff as compared to soft gels (see Figure 4D). 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 (see Figure 4E). However, the invasion efficiency (bacteria internalized/total number of bacteria) of Lm into HMEC-1 cells is similar, irrespective of substrate stiffness (see Figure 4F).
Traction force microscopy of Lm-infected HMEC-1 cells: 
Lm infection of HMEC-1 cells could alter the biomechanics of infected host cells, including the strength of attachment to their ECM or to each other, affecting their barrier integrity. We sought to evaluate whether that could be the case by using Traction Force Microscopy32 to calculate the cell-ECM traction forces and Monolayer Stress Microscopy41 to calculate the intracellular tensional forces. Figure 5 depicts maps of the deformation field, traction stress field, and intracellular tension field of HMEC-1 cells residing on 20-kPa hydrogels at different time points post-infection. Figures 5A - 5D refer to uninfected control HMEC-1 cells while Figures 5E - 5H refer to HMEC-1 cells infected with Lm at a multiplicity of infection equal to 300 bacteria/cell. This preliminary work suggests that infected HMEC-1 cells reduce the magnitude of their cell-ECM and intracellular stresses during the course of an infection with Lm, whereas that is not observed for uninfected control cells.
ECM stiffness-dependent Lm dissemination across HMEC-1 monolayers: 
Time-lapse microscopy was used to investigate the effect matrix stiffness has on Lm dissemination through HMEC-1 monolayers. As Lm spreads through the monolayer, the bacteria create a focus of infection that grows as a function of time (see Figure 6A and Video Figures 1 and 2). The area of the infection focus was measured by drawing a convex hull, the smallest convex polygon that encompasses a set of points, around the bacteria42. There is no standard metric in the field to measure the efficiency of L. monocytogenes cell-to-cell spread through a monolayer of host cells. To assess spread efficiency, some have counted the number of host cells in an infection focus41, and others have drawn boundaries manually around the group of infection host cells43. We chose to draw a convex polygon around the bacteria, because it is an automated, consistent, and computationally inexpensive process to measure the efficiency of L. monocytogenes spread. By doing so, we found a slight decrease in the infection focus area in the HMEC-1 cells seeded onto 70 kPa hydrogels when compared to those seeded on 3 kPa matrices (see Figure 6B and Video Figures 1 and 2). To determine the rate of growth of the infection focus, the radial distance was plotted as a function of time by taking the square root of the area of the infection focus and dividing this by pi. This mathematical transformation assumes that the shape of the infection focus is roughly circular44. To measure the speed of the focus growth, the rate of change (i.e., the slope) of the radial distance for both 3- and 70-kPa matrices was measured. This approach elucidated that the infection focus grew faster and monotonically in HMEC-1 cells seeded onto 3-kPa matrices. However, the focus grew significantly slower (first 200 min) and slightly slower (200 to 600 min) in cells seeded onto 70-kPa matrices (see Figure 6C). Indeed, analysis of further data confirmed that the infection focus grew, on average, two-fold slower in 70-kPa matrices, especially during the first 200 min (see Figure 6D).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57361/57361fig3.jpg" /> 
Figure 3: ECM stiffness-dependent susceptibility of HMEC-1 cells to Lm invasion measured by using flow cytometry.&#160;HMEC-1 cells were infected with Lm (JAT985) and the infection was analyzed by flow cytometry. A. This panel shows a side versus forward scatter plot for representative HMEC-1 cells coming from a single well. The bulk distribution of cells was selected via gating to exclude debris (left) and cell doublets or triplets (right). B. This graph shows a histogram of the logarithm of the Lm fluorescence intensity per cell for HMEC-1 plated on soft 0.6 kPa. C. This graph shows a histogram of the logarithm of the Lm fluorescence intensity per cell for HMEC-1 plated on stiff 70 kPa hydrogels. The histograms for N = 4 - 6 replicates are shown in different colors. The control uninfected cells&#39; histogram is purple. The gate used to define what is infected is shown in red. The MOI is 100 and the infection was assessed 8 h post-infection. D. Data show percentage of infected HMEC-1 cells versus hydrogel stiffness (N = 5). The horizontal bars depict the data&#39;s mean. The P-value was calculated with the non-parametric Wilcoxon Rank Sum test. <a href="/files/ftp_upload/57361/57361fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video Figure 1" src="/files/ftp_upload/57361/57361mov1.jpg" /> 
Video Figure 1: Dissemination of intracellular Lm (red channel) through an HMEC-1 cell monolayer (phase) seeded on a 3-kPa substrate. The cells were imaged in Leibovitz&#39;s L-15 media (10% FBS, 20 &#181;g/mL of gentamicin) inside an environmental chamber equilibrated to 37 &#730;C. The images were collected every 5 min. The movie speed is 15 frames/s. <a href="https://www.jove.com/files/ftp_upload/57361/Video_Figure_1.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Video Figure 2" src="/files/ftp_upload/57361/57361mov2.jpg" /> 
Video Figure 2: Dissemination of intracellular Lm (red channel) through an HMEC-1 cell monolayer (phase) seeded on a 70-kPa substrate. The cells were imaged in Leibovitz&#39;s L-15 media (10% FBS, 20 &#181;g/mL of gentamicin) inside an environmental chamber equilibrated to 37 &#176;C. The images were collected every 5 min. The movie speed is 15 frames/s. <a href="https://www.jove.com/files/ftp_upload/57361/Video_Figure_2.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Young&#8217;s modulus (E, kPa)</td>
			<td>Acrylamide % (from 40% stock)</td>
			<td>Bisacrylamide % (from 2% stock)</td>
		</tr>
		<tr>
			<td>0.6</td>
			<td>3</td>
			<td>0.045</td>
		</tr>
		<tr>
			<td>3</td>
			<td>5</td>
			<td>0.075</td>
		</tr>
		<tr>
			<td>10</td>
			<td>10</td>
			<td>0.075</td>
		</tr>
		<tr>
			<td>20</td>
			<td>8</td>
			<td>0.195</td>
		</tr>
		<tr>
			<td>70</td>
			<td>10</td>
			<td>0.45</td>
		</tr>
	</tbody>
</table>
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&#39;s modulus, E) are indicated in different columns.
<img alt="Supplemental Material" src="/files/ftp_upload/57361/57361supfig1.jpg" /> 
Supplemental Material 1. Computation of intracellular tension from calculated traction stresses. <a href="https://www.jove.com/files/ftp_upload/57361/Supplemental_Material_1.docx" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells at JoVE.

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

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