Name: Serie: Ein Assay zur Untersuchung der Rolle von Vitronectin bei der bakteriellen Adhärenz an Epithelzellen des Wirts - Konzept
Uploaded: 2023-09-29T11:21:26.000+00:00
Duration: 1 min 55 s
Description: 59 Aufrufe. Quelle: Singh, B. et al., Assays zur Untersuchung der Rolle von Vitronectin bei bakterieller Adhäsion und Serumresistenz. J. Vis. Exp. (2018) In diesem Video demonstrieren wir einen Assay, um die Rolle von Vitronektin bei der Adhärenz von Haemophilus influenzae an den Wirtsepithelzellen zu untersuchen.

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1.&#160;Study of Vn-dependent adherence of bacteria to epithelial cells
<ol>
	<li>Culture A549 cells (type II alveolar epithelial cells) in a 75-cm2&#160;tissue culture flask with F12 medium supplemented with 10% (vol/vol) fetal calf serum (FCS) (complete medium) and 5 &#181;g/mL gentamicin. Incubate the flask in an incubator with 5% CO2&#160;at 37 &#176;C for 3 days until 80% confluent (confluency can be estimated by visualizing surface coverage by the cells using an inverted microscope). The following four steps describe how to prepare the epithelial cells for the assay.
	<ol>
		<li>Wash the cell monolayer in the flask twice with 20 mL of PBS by gentle swirling. To detach the cells from the flask surface, add 2 mL of cell detachment enzyme solution and incubate the flask for 5 min at 37 &#176;C. Tap the flask with your palm to detach all the cells from the plastic surface. Pipette up and down a few times to disperse cell clumps.</li>
		<li>Add 18 mL of F12 complete medium to the flask and transfer the entire cell suspension to a 50-mL sterile Falcon tube. Centrifuge the cell suspension for 5 min at 200 x g at RT and discard the supernatant. Resuspend the cell pellet in 10 mL of F12 complete medium.</li>
		<li>Remove 10 &#181;L of the cell suspension and place it in an Eppendorf tube, then add 90 &#181;L of Trypan blue solution. Load the sample into a hemocytometer (depth 0.1 mm) after placing the coverslip.</li>
		<li>Count all viable cells in areas A, B, C, and D (each field consists of 16 squares, and each square has an area of 0.0025 mm2), then calculate the average number of cells ([A+B+C+D]/4). Calculate the number of cells per milliliter using the following equation: 
		Viable cells/mL = average cell count &#215; 104 &#215; dilution factor. 
		NOTE: Here, the dilution factor is 10.</li>
		<li>Dilute the cell suspension to 5.0 x 103 cells/mL using a complete medium containing 5 &#181;g/mL gentamicin. Dispense 500 &#956;L of cell suspension into each well of a 24-well cell culture plate. Incubate the plate at 37 &#176;C in 5% CO2 until the cells are 90% confluent.</li>
	</ol>
	</li>
	<li>Prior to bacterial infection, remove the medium from the wells and add F12 medium (without FCS), then incubate overnight at 37 &#176;C.</li>
	<li>Wash the cell monolayer three times with 500 &#181;L of PBS at RT. Place the plate on ice and add 100 &#181;L of pre-chilled F12 medium containing 10 &#181;g/mL of Vn. Incubate plate at 4 &#176;C for 1 h. For control wells, add only F12 medium.</li>
	<li>After incubation, discard the solution by pipetting and wash the cell layer twice with 1 mL of PBS at RT.</li>
	<li>Culture Hif clinical isolates in brain-heart infusion (BHI) medium supplemented with 10 &#181;g/mL NAD and hemin at 37 &#176;C with shaking at 200 rpm. Use Luria-Bertani medium to culture E. coli. Harvest Hif at mid-log phase (OD600 = 0.3) and resuspend the bacteria in phosphate-buffered saline (PBS), pH 7.2, containing 1% (w/v) bovine serum albumin (BSA) (blocking buffer; PBS-BSA). Adjust the suspension to 109 CFU/mL. Resuspend a culture of freshly grown Hif M10 in F12 medium (2&#215;108 CFU/mL). Add 100 &#181;L of this bacterial suspension to each well and incubate the plate for 2 h at 37 &#176;C. 
	NOTE: Each well contains approximately 2 x 105 A549 cells. For infection, 2 x 107 bacterial CFU were added, corresponding to a multiplicity of infection of 100.</li>
	<li>Remove the medium by pipetting and wash the A549 epithelial cells three times with PBS. Add 50 &#181;L/well of cell detachment solution and incubate the plate for 5 min at 37 &#176;C.</li>
	<li>Next, add 50 &#181;L of F12 complete medium per well to stop the enzymatic reaction. Transfer the epithelial cells (&#8776;100 &#956;L [i.e., the entire volume]) from each well to a 6-mL glass tube containing four glass beads. Lyse the cells at RT by vortexing for 2 min.</li>
	<li>Dilute 10 &#181;L of the cell lysate 100-fold by adding 10 &#181;L of lysate to 990 &#181;L of F12 medium. Plate 10 &#181;L of the diluted sample onto a chocolate agar plate.</li>
	<li>Incubate the chocolate agar plate at 37 &#176;C overnight, then count the colonies. Each colony represents one CFU.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5 mL polystyrene round-bottom tube</td>
			<td>BD Falcon</td>
			<td>60819-138</td>
			<td>12 &#215; 75 mm style</td>
		</tr>
		<tr>
			<td>5% CO2 supplied incubator</td>
			<td>Thermo Scientific</td>
			<td>BBD6220</td>
			<td></td>
		</tr>
		<tr>
			<td>6 mL polystyrene round-bottom tube</td>
			<td>VWR</td>
			<td>89000-478</td>
			<td>12 &#215; 75 mm style with cap</td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>BD Falcon</td>
			<td>08-772-1H</td>
			<td>Cell culture grade</td>
		</tr>
		<tr>
			<td>75 cm2 tissue culture flask</td>
			<td>BD Falcon</td>
			<td>BD353136</td>
			<td>Vented</td>
		</tr>
		<tr>
			<td>A549 Cell Line human</td>
			<td>Sigma-Aldrich</td>
			<td>86012804-1VL</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell detachment enzyme (Accutase)</td>
			<td>Sigma-Aldrich</td>
			<td>A6964-500ML</td>
			<td>Cell Culture Grade</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumins (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A2058</td>
			<td>Suitable for cell culture</td>
		</tr>
		<tr>
			<td>F12 medium</td>
			<td>Sigma-Aldrich</td>
			<td>D6421</td>
			<td>Cell Culture Grade</td>
		</tr>
		<tr>
			<td>Fetal Calf Serum (FCS)</td>
			<td>Sigma-Aldrich</td>
			<td>12003C</td>
			<td>Suitable for cell culture</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Marienfeld</td>
			<td>640210</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture flask</td>
			<td>BD Falcon</td>
			<td>3175167</td>
			<td>75 cm2</td>
		</tr>
		<tr>
			<td>Vitronectin (Vn) from human plasma</td>
			<td>Sigma-Aldrich</td>
			<td>V8379-50UG</td>
			<td>Cell culture grade</td>
		</tr>
	</tbody>
</table>

an assay to study the role of vitronectin in bacterial adherence to host epithelial cells

Source: Singh, B. et al., <a href="https://app.jove.com/v/54653">Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance.</a> J. Vis. Exp. (2018)
In this video, we demonstrate an assay to study the role of vitronectin in Haemophilus influenzae adherence to the host epithelial cells.

An Assay to Study the Role of Vitronectin in Bacterial Adherence to Host Epithelial Cells

Source: Singh, B. et al., Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance. J. Vis. Exp. (2018)
In this video, we ...

Take a multi-well plate containing adherent epithelial cell monolayers. Replace media with serum-free media, preventing serum protein interference. Post-incubation, remove media. Wash the cells with buffer. Add chilled serum-free media containing vitronectin. Incubate at lower temperatures.
The N-terminal RGD-domain of vitronectin binds to the epithelial cell surface integrin receptors. Lower temperatures inhibit cellular internalization of the vitronectin-integrin complex.
Following integrin receptor saturation, remove media. Wash with buffer to remove excess vitronectin, which could impact the bacterial vitronectin-dependent integrin binding.
Add Haemophilus influenzae type f, or Hif, a gram-negative bacterial pathogen, suspension. Incubate.
Protein H on the Hif surface binds to the vitronectin&#39;s C-terminus, facilitating Hif adherence to the epithelial cell and subsequent internalization.
Remove media and wash with buffer to remove unbound Hif. Use a cell detachment enzyme to harvest the cells. Add serum-containing media to stop the enzymatic reaction. Transfer the cell suspension to a glass tube containing glass beads. Vortex to lyse the cells, releasing internalized and surface-attached bacteria.
Dilute the lysate with serum-free media and seed on a chocolate agar plate. Incubate for Hif to form colonies. Count the colonies.
A higher number of colonies indicates enhanced Hif adherence to the epithelial cells in the presence of vitronectin.

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An Assay to Study the Role of Vitronectin in Bacterial Adherence to Host Epithelial Cells

Transkript

An Assay to Study the Role of Vitronectin in Bacterial Adherence to Host Epithelial Cells