an assay to study the impact of extracellular matrix stiffness on bacterial infection

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.

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1. Manufacturing Thin Two-layered Polyacrylamide (PA) Hydrogels on Multi-Well Plates
<ol>
	<li>Polyacrylamide hydrogel fabrication 
	NOTE: See&#160;Figure 1.
	<ol>
		<li>Prepare aqueous solutions that contain 3 - 10% of 40% stock acrylamide solution (see&#160;Table of Materials) and 0.06 - 0.6% of 2% stock bis-acrylamide solution (see&#160;Table of Materials) to manufacture hydrogels of tunable stiffness ranging from 0.6 kPa to 70 kPa. See&#160;Table 1.
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>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-&#181;m diameter fluorescent micro-beads (see&#160;Table of Materials).</li>
		<li>Degas Solutions 1 and 2 by vacuum for 15 min to eliminate oxygen that is known to inhibit polymerization of the solutions.</li>
		<li>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.</li>
		<li>Add 3.6 &#181;L of Solution 1 to the center of each well of the 24-well dish.</li>
		<li>Immediately cover the wells with 12-mm untreated circular coverslips and let Solution 1 sit for 20 min so that it fully polymerizes.</li>
		<li>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.</li>
		<li>Add 0.6% of the 10 g/mL stock APS solution and 0.43% TEMED to Solution 2. Deposit 2.4 &#181;L of the mixture on top of the first layer in each well of the 24-well dish.</li>
		<li>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.</li>
		<li>Add 500 &#181;L of 50 mM HEPES pH 7.5 to each of the wells and then remove the glass coverslips with the syringe needle and forceps.</li>
	</ol>
	</li>
	<li>Sterilization, collagen-coating, and equilibration of polyacrylamide hydrogels
	<ol>
		<li>UV-expose the hydrogels for 1 h in the tissue culture hood to allow sterilization.</li>
		<li>Prepare a mixture of 0.5% weight/volume sulfosuccinimidyl 6-(4&#8242;-azido-2&#8242;-nitrophenylamino)hexanoate (Sulfo-SANPAH, see&#160;Table of Materials) in 1% DMSO and 50 mM HEPES pH = 7.5.</li>
		<li>Add 200 &#181;L of this solution to the upper surface of the hydrogels. Acting fast, expose them to UV (302 nm) for 10 min to activate them.</li>
		<li>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.</li>
		<li>Protein-coat the hydrogels with 200 &#181;L of 0.25 mg/mL rat tail Collagen I (see&#160;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.</li>
		<li>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&#39; 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 measurements (AFM) can also be performed at this point to confirm the exact stiffness of the hydrogels (see&#160;Figure 2).</li>
		<li>Before seeding the cells of interest on the hydrogels, add 1 mL of media on the hydrogels and incubate them at 37 &#730;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&#160;step 2.1&#160;for details).</li>
	</ol>
	</li>
</ol>
2. Human Microvascular Endothelial Cell Culture and Seeding on Hydrogels
<ol>
	<li>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 &#181;g/mL of hydrocortisone, and 2 mM of L-glutamine (see&#160;Table of Materials).</li>
	<li>Split the confluent cultures 1:6 every 3 - 4 days and keep the cells until passage 40.</li>
	<li>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 &#176;C for 5 - 10 min to allow the detachment of the cells from their substrate.</li>
	<li>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.</li>
	<li>Gently swirl the solution of cells to ensure that the cells are evenly distributed and then take out 20 &#181;L of the solution and very gently fill out the two chambers underneath the coverslip of a glass hemocytometer.</li>
	<li>Pellet down the solution of cells contained in the conical centrifuge tube using centrifugation for 10 min at 500 x g.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#160;cells/mL.</li>
	<li>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.</li>
</ol>
3. Infection of Human Microvascular Endothelial Cells with&#160;L. monocytogenes
<ol>
	<li>Three days before the infection, streak out the Lm strain to be used from a glycerol stock (stored at -80 &#176;C) onto a BHI-agar plate that contains 7.5 &#181;g/mL of chloramphenicol and 200 &#181;g/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).</li>
	<li>Incubate plates at 37 &#176;C until discrete colonies are formed (1 - 2 days).</li>
	<li>The day before the infection, grow the desired strain overnight, shaking it at 150 rpm at 30 &#176;C in BHI media with 7.5 &#181;g/mL of chloramphenicol (if appropriate).
	<ol>
		<li>Place 5 mL of BHI media in a 15-mL conical centrifuge tube, add 7.5 &#181;g/mL of chloramphenicol (if appropriate), and then inoculate a single colony from the agar plate using a sterile 10 &#181;L tip.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Dilute the overnight culture to an OD600 of 0.1 and incubate it, shaking for 2 h at 30 &#176;C, in BHI media with 7.5 &#181;g/mL of chloramphenicol (if appropriate) to allow the bacteria to reach log-phase growth.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Prepare the infection mix by mixing 10 or 50 &#181;L of the bacteria resuspended in PBS with 1 mL of MCDB-131 full media for a multiplicity of infection (MOI;&#160;i.e., the number of bacteria per host cell) of approximately 50 bacteria per host cell or 10 bacteria per host cell.</li>
	<li>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.</li>
	<li>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 &#176;C.</li>
	<li>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 &#181;g/mL of gentamicin.</li>
</ol>
4. Flow Cytometry to Quantify Extracellular-matrix-stiffness Dependent Susceptibility of Host Cells to Infection
<ol>
	<li>Weigh 5 mg of collagenase and place it in a 15-mL conical centrifuge tube. Add 10 mL of 0.25% trypsin-EDTA and mix them well.</li>
	<li>8 h post-infection, remove the media from the wells of the 24-well plate and wash the wells 1x with tissue culture PBS. Remove the PBS from the wells and add 200 &#181;L of the trypsin-EDTA/collagenase mix to each well. Place this in the tissue culture incubator for 10 min to allow full detachment of the cells.</li>
	<li>Use a fresh pipette for each well and pipet the mix up and down 8x. Be gentle so as to not damage the cells. Add 200 &#181;L of full media to each well to neutralize the trypsin.</li>
	<li>Transfer the 400-&#181;L cell solution of each well into a 5-mL polystyrene tube with a 35-&#181;m cell strainer cap (see&#160;Table of Materials).</li>
	<li>Analyze 10,000 - 20,000 cells per sample by flow cytometry. Perform data acquisition through flow cytometry and determine the percentage of infected cells per well using relevant commercially available software. Use the forward scatter versus side scatter plots to gate the bulk of the distribution of the cell counts. 
	NOTE:: This will ensure the analysis of single cells and eliminate debris or cell doublets or triplets.
	<ol>
		<li>Measure the fluorescence signal from control-uninfected cells and gate the population of infected cells excluding autofluorescence.</li>
	</ol>
	</li>
</ol>
Table 1. Composition of polyacrylamide (PA) hydrogels of varying stiffness.&#160;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.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Young&#39;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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide pellets</td>
			<td>Fisher</td>
			<td>S318-500</td>
			<td></td>
		</tr>
		<tr>
			<td>(3-Aminopropyl)triethoxysilane</td>
			<td>Sigma</td>
			<td>A3648</td>
			<td></td>
		</tr>
		<tr>
			<td>25% gluteraldehyde</td>
			<td>Sigma</td>
			<td>G6257-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>40% Acrylamide</td>
			<td>Sigma</td>
			<td>A4058-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bis-acrylamide solution (2%w/v)</td>
			<td>Fisher Scientific</td>
			<td>BP1404-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorospheres carboxylate-modified microspheres, 0.1 &#956;m, yellow-green fluorescent (505/515)</td>
			<td>Invitrogen</td>
			<td>F8803</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Persulfate</td>
			<td>Fisher</td>
			<td>BP17925</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Sigma</td>
			<td>T9281-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-SANPAH</td>
			<td>Proteochem</td>
			<td>c1111-100mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen, Type I Solution from rat</td>
			<td>Sigma</td>
			<td>C3867-1VL</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>J.T. Baker</td>
			<td>9224-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES, Free acid</td>
			<td>J.T. Baker</td>
			<td>4018-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 medium, no phenol red</td>
			<td>Thermofischer</td>
			<td>21083027</td>
			<td></td>
		</tr>
		<tr>
			<td>MCDB 131 Medium, no glutamine</td>
			<td>Life technologies</td>
			<td>10372019</td>
			<td></td>
		</tr>
		<tr>
			<td>Foundation Fetal Bovine Serum, Lot: A37C48A</td>
			<td>Gemini Bio-Prod</td>
			<td>900108 500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor, EGF</td>
			<td>Sigma</td>
			<td>E9644</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma</td>
			<td>H0888</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 200mM</td>
			<td>Fisher</td>
			<td>SH3003401</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS 1X</td>
			<td>Fisher</td>
			<td>SH30028FS</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin sulfate</td>
			<td>MP biomedicals</td>
			<td>194530</td>
			<td></td>
		</tr>
		<tr>
			<td>Cloramphenicol</td>
			<td>Sigma</td>
			<td>C0378-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>DifcoTM Agar, Granulated</td>
			<td>BD</td>
			<td>214530</td>
			<td></td>
		</tr>
		<tr>
			<td>BBL TM Brain-heart infusion</td>
			<td>BD</td>
			<td>211059</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, trihydrochloride, trihydrate - 10 mg/ul solution in water</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde 16% EM grade</td>
			<td>Electron microscopy</td>
			<td>15710-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Listeria monocytogenes antibody</td>
			<td>Abcam</td>
			<td>ab35132</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 546 conjugate</td>
			<td>Thermofischer</td>
			<td>A-11035</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% trypsin-EDTA , phenol red</td>
			<td>Thermofischer</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>COLLAGENASE FROM CLOSTRIDIUM HISTOLYTIC</td>
			<td>Sigma</td>
			<td>C8051</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin sulfate</td>
			<td>Fisher Scientific</td>
			<td>3810-74-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Calbiochem</td>
			<td>8510</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Thermofischer</td>
			<td>28364</td>
			<td></td>
		</tr>
		<tr>
			<td>MES powder</td>
			<td>Sigma</td>
			<td>M3885</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>J.T. Baker</td>
			<td>3040-05</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>J.T. Baker</td>
			<td>2444-1</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Acros</td>
			<td>40991</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable lab equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm circular glass coverslips</td>
			<td>Fisherbrand</td>
			<td>12-545-81</td>
			<td>No. 1.5 Coverslip | 10 mm Glass Diameter | Uncoated</td>
		</tr>
		<tr>
			<td>Glass bottom 24 well plates</td>
			<td>Mattek</td>
			<td>P24G-1.5-13-F</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml polystyrene tubes with a 35 &#956;m cell strainer cap</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>T-25 flasks</td>
			<td>Falcon</td>
			<td>353118</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conicals tubes</td>
			<td>Falcon</td>
			<td>352196</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Serological Pipettes (1 ml, 2 ml, 5 ml, 10 ml, 25 ml)</td>
			<td>Falcon</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur Glass Pipettes</td>
			<td>VWR</td>
			<td>14672-380</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips (1-200 &#956;l, 101-1000 &#956;l)</td>
			<td>Denville</td>
			<td>P1122, P1126</td>
			<td></td>
		</tr>
		<tr>
			<td>Powder Free Examination Gloves</td>
			<td>Microflex</td>
			<td>XC-310</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvettes bacteria</td>
			<td>Sarstedt</td>
			<td>67.746</td>
			<td></td>
		</tr>
		<tr>
			<td>Razors</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe needle</td>
			<td>BD</td>
			<td>305167</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2um sterilizng bottles</td>
			<td>Thermo Scientific</td>
			<td>566-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>20 ml syringes</td>
			<td>BD</td>
			<td>302830</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2um filters</td>
			<td>Thermo Scientific</td>
			<td>723-2520</td>
			<td></td>
		</tr>
		<tr>
			<td>wooden sticks</td>
			<td>Grainger</td>
			<td>42181501</td>
			<td></td>
		</tr>
		<tr>
			<td>Saran wrap</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-3687</td>
			<td></td>
		</tr>
		<tr>
			<td>Plates bacteria</td>
			<td>Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Large/non-disposable lab equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Hood</td>
			<td>Baker</td>
			<td>SG504</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Sigma</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteria incubator</td>
			<td>Thermo Scientific</td>
			<td>IGS180</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture Incubator</td>
			<td>NuAire</td>
			<td>NU-8700</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum chamber/degasser</td>
			<td>Belart</td>
			<td>42025</td>
			<td>37 &#176;C and 5% CO2</td>
		</tr>
		<tr>
			<td>Inverted Nikon Diaphot 200 epifluorescence microscope</td>
			<td>Nikon</td>
			<td>NIKON-DIAPHOT-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Cage Incubator</td>
			<td>Haison</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanford FACScan analyzer</td>
			<td>Stanford and Cytek upgraded FACScan</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Aid</td>
			<td>Drummond</td>
			<td>4-000-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettors (10 &#956;l, 200 &#956;l, 1000ul)</td>
			<td>Gilson</td>
			<td>F144802, F123601, F123602</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Mettler Toledo</td>
			<td>30019028</td>
			<td></td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>FST</td>
			<td>11000-12</td>
			<td></td>
		</tr>
		<tr>
			<td>1 L flask</td>
			<td>Fisherbrand</td>
			<td>FB5011000</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave machine</td>
			<td>Amsco</td>
			<td>3021</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir magnet plate</td>
			<td>Bellco</td>
			<td>7760-06000</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnet stirring bars</td>
			<td>Bellco</td>
			<td>1975-00100</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Beckman</td>
			<td>DU 640</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanford FACScan analyzer</td>
			<td>Cytek Biosciences</td>
			<td>Custom Stanford and Cytek upgraded FACScan</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Software (&#956;Manager)</td>
			<td>Open Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Matlab Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flowjo</td>
			<td>FlowJo, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Automated image analysis software, CellC</td>
			<td>https://sites.google.com/site/cellcsoftware/</td>
			<td></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>

Source: Bastounis, E. E., et al. <a href="https://app.jove.com/v/57361">A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells.</a> J. Vis. Exp. (2018).
This video demonstrates an assay to study the effect of extracellular matrix (ECM) stiffness on bacterial infection of adherent cells. Polyacrylamide hydrogels of varying stiffness, overlaid with collagen, are layered in a multi-well plate to mimic the stiffness of physiological ECM. Upon overlaying human endothelial cells on the hydrogels and infecting the cells with Listeria monocytogenes bacteria engineered to express a fluorescent protein once intracellular, flow cytometry is used to compare internalized bacteria in cells residing on stiffer hydrogels versus softer hydrogels.

<img alt="Figure 1" src="/files/ftp_upload/22005/22005_Figure1.jpg" /> 
Figure 1: Bacterial infection assay of host cells residing on thin two-layered fluorescent bead-embedded polyacrylamide (PA) hydrogels of varying stiffness. A.&#160;Glass coverslips are chemically modified to enable hydrogel attachment.&#160;B. 3.6 &#181;L of PA mixtures are deposited on the glass bottoms.&#160;C. The mixture is covered with a 12-mm circular glas...

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

	Polyacrylamide hydrogel fabrication
	NOTE: See&#160;Figure 1.
	
		...

Take wells containing polyacrylamide hydrogels of varying stiffness overlaid with collagen, mimicking a physiological extracellular matrix, or ECM.
Add microvascular endothelial cells that form focal adhesions, or FAs &#8212; complexes mediating cell-ECM binding &#8212; to sense the hydrogel stiffness.
Stiffer hydrogels elevate cell-surface expression of vimentin, a cytoskeletal component, compared to softer hydrogels.
Add Listeria monocytogenes bacteria, engineered to express a fluorescent protein under the promoter for actin assembly-inducing protein, or ActA.
Centrifuge to synchronize bacterial contact with the host cells, and incubate.
The bacteria bind to vimentin. More vimentin on the stiffer hydrogel-bound host cells leads to more bacterial adherence and endocytosis.
Add an antibiotic, eliminating non-internalized bacteria.
The internalized bacteria secrete toxins, rupturing the endosome membrane. Cytoplasmic escape activates the ActA promoter to express the fluorescent protein, labeling intracellular bacteria.
Enzymatically detach the cells. Using flow cytometry, detect a higher fluorescence in host cells residing on stiffer hydrogels, indicating greater bacterial internalization.

11 조회수. 세포외 기질 강성이 박테리아 감염에 미치는 영향을 연구하기 위한 분석

비디오: 세포외 기질 강성이 박테리아 감염에 미치는 영향을 연구하기 위한 분석 - 실험

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

신경 문장 세포에서 기계적 신호를 연구하기 위한 최적화된 O9-1/하이드로겔 시스템

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...

연구

JoVE Journal

발생학

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

폴 리 비닐 알코올-Co-Itaconic 산 Hydrogels 이종 없는 조건 하에서 다양 한 강성에 인간의 Pluripotent 줄기 세포 문화

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

생체공학

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Widefield 형광 현미경을 사용 하 여 Mechanobiology 연구에 대 한 부드러운 실리콘 기판의 강성 측정

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

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

내레이션 대본

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