This work was supported by National Institutes of Health (NIH) grants R01-DK080752 and P50-DK064540. We thank J. Loughman for her efforts in establishing this assay.

1. Culturing 5637 Bladder Epithelial Cells
Perform the following steps in a laminar flow tissue-culture hood prepared with UV irradiation and wiped down with 70% ethanol.
<ol>
	<li>Prepare RPMI-1640 medium containing 10% fetal bovine serum (FBS) [termed RPMI+], filter sterilize using a 0.22 &#181;m pore size filter, and warm to 37 &#176;C in a water bath.</li>
	<li>Thaw a cryovial of 5637 cells (American Type Culture Collection HTB-9; derived from bladder carcinoma) in a 37 &#176;C water bath. Quickly transfer the thawed cells to a 75 cm2 tissue culture flask containing 20 ml RPMI+.</li>
	<li>Incubate the flask at 37 &#176;C in a humidified atmosphere with 5% CO2 until the cells are approximately 95% confluent (~6 x 106 cells per flask), about 4 days.</li>
	<li>To subculture the 5637 cells:
	<ol>
		<li>Remove the medium from the flask. Wash the cells with 10 ml Dulbecco&#39;s Phosphate-Buffered Saline (DPBS) at room temperature.</li>
		<li>Add 6 ml warm (37 &#176;C) 0.05% trypsin, 0.02% EDTA solution to the flask, and incubate at 37 &#176;C with 5% CO2 for 15 min.</li>
		<li>Transfer the cells to a 15 ml conical tube, and pellet by centrifugation at 300 x g for 5 min. Remove the trypsin/EDTA solution.</li>
		<li>Resuspend the cells in 6 ml RPMI+ (106 cells/ml), and transfer 1 ml (106 cells) to a new 75 cm2 flask containing 20 ml RPMI+.</li>
	</ol>
	</li>
	<li>Repeat step 1.4 to subculture cells every 4 days or when the cells reach 95% confluence.</li>
</ol>
2. Seeding and Growing 5637 Cells on Permeable Supports
Perform the following steps in a tissue culture hood using aseptic technique. For optimal results, use 5637 cells that have undergone fewer than 10 subcultures.
<ol>
	<li>Using sterile forceps, invert the permeable supports in a sterile 25 mm deep tissue culture dish.</li>
	<li>Trypsinize a 75 cm2 flask of 5637 cells at 95% confluence according to steps 1.4.1-1.4.3.</li>
	<li>Using a 1,000 &#181;l pipette, gently resuspend the cells in ~2 ml RPMI+ to a concentration of 3 x 106 cells/ml, determined by cell counting using a hemocytometer.</li>
	<li>Apply 50 &#181;l of the cell suspension (1.5 x 105 cells) to each permeable support without touching the membrane. Place the lid on the dish, and carefully place the dish at 37 &#176;C with 5% CO2 for no more than 16 hr.</li>
	<li>Using sterile forceps, right the permeable supports into a 24-well plate containing 0.6 ml RPMI+ per well. Add 0.1 ml RPMI+ to the upper reservoir of each permeable support. Incubate at 37 &#176;C with 5% CO2.</li>
	<li>Replace the medium every 2 days. First, aspirate medium from the upper reservoir followed by the lower reservoir, and then apply fresh RPMI+ in the reverse order, to the lower reservoir (0.6 ml) and then the upper reservoir (0.1 ml).</li>
	<li>Seven days after seeding the permeable supports with 5637 cells, assess confluence of the cells. Fill the upper reservoir with RPMI+ (~0.35 ml). The cells are sufficiently confluent when the medium does not equilibrate between the upper and lower reservoirs.</li>
</ol>
3. Preparation of the Bacterial Inoculum
<ol>
	<li>Using aseptic technique, add 20 ml of appropriate culture broth to a 250 ml flask with a cap. Use Luria-Bertani (LB) broth for E. coli cultures, and add antibiotics to the broth where appropriate.</li>
	<li>Using a sterile inoculation loop, inoculate the broth with bacteria from a glycerol stock or a streak plate. For UPEC strains, incubate the bacterial culture at 37 &#176;C for approximately 16 hr, without shaking (to promote production of type 1 pili).</li>
	<li>Transfer the bacterial culture to a centrifuge tube. Pellet the bacteria by centrifugation at 8,000 x g for 10 min.</li>
	<li>Decant the supernatant, and resuspend the bacteria in PBS to OD600 = 1.000, equivalent to ~109 CFU/ml.
	<ol>
		<li>To generate a heat-killed bacterial stimulus, incubate an aliquot (e.g.&#160;~1.5 x 108 CFU in 150 &#181;l) of the resuspended bacteria at 55 &#176;C for 30 min. Plate an aliquot of the heat-killed suspension to confirm bacterial death.</li>
	</ol>
	</li>
	<li>Immediately before use, dilute the resuspended bacteria (live or heat-killed) 10-fold to 108 CFU/ml in warm serum-free RPMI [termed RPMI-] in a microfuge tube.</li>
</ol>
4. Isolation of Human Neutrophils from Peripheral Blood
The collection of blood from adult volunteers requires advance review and approval from an institutional review board. Wear appropriate personal protective equipment and properly dispose of hazardous materials to avoid exposure to human blood.
<ol>
	<li>Approximately 25 ml of venous blood from a healthy adult volunteer should be drawn into 3 sterile sodium heparin-containing blood collection tubes by staff trained in phlebotomy.
	<ol>
		<li>Tightly wrap a rubber tourniquet around the upper arm, above the elbow.</li>
		<li>Disinfect the entry site, the antecubital fossa, using a sterile 70% alcohol wipe.</li>
		<li>Attach a plastic tube holder to a winged butterfly needle system.</li>
		<li>Insert the needle into an antecubital vein at a 30&#176; angle or less, bevel facing up. The needle has punctured the vein when a spurt of blood appears in the plastic tubing.</li>
		<li>Insert a blood collection tube into the plastic tube holder. When the first collection tube is full, replace with the second collection tube. Repeat with the third tube.</li>
		<li>When the third collection tube is approximately half full, remove the tourniquet.</li>
		<li>Cover the puncture site with a sterile cotton ball and slowly withdraw the needle from the vein. Slide the protective shield over the needle and place in a biohazard sharps container.</li>
		<li>Apply pressure to the puncture site. Cover the puncture site and cotton ball with an adhesive bandage.</li>
		<li>Gently invert the blood collection tubes to disperse the heparin.</li>
	</ol>
	</li>
</ol>
Perform the following steps in a tissue culture hood using aseptic technique.
<ol start="2">
	<li>Using a 10 ml pipette, gently transfer the blood to a fresh, sterile 50 ml conical tube. Add an equivalent volume of 3% (w/v) dextran in 0.9% NaCl and mix by inversion. Incubate the tube upright at room temperature for 20 min.</li>
	<li>Without disrupting the lower layer, carefully aspirate the upper layer and transfer it to a new 50 ml conical tube. Pellet the cells by centrifugation at 300 x g for 10 min. Discard the supernatant.</li>
	<li>Resuspend the cell pellet in a volume of 0.9% NaCl equivalent to the starting volume of blood.</li>
	<li>Layer 10 ml of density centrifugation solution under the cell suspension, preserving the interface between the two phases. Centrifuge at 400 x g for 30 min with no brake. Discard the supernatant.</li>
	<li>To lyse remaining red blood cells, resuspend the cell pellet in 10 ml cold 0.2% NaCl. Incubate for 30 sec, and then promptly add 10 ml cold 1.6% NaCl to restore isotonicity.</li>
	<li>Pellet the cells by centrifugation at 300 x g for 6 min. Discard the supernatant.</li>
	<li>Repeat steps 4.6-4.7 until the cell pellet appears to be free of red blood cells, typically 3 rounds of lysis.</li>
	<li>Using a 1,000 &#181;l pipette, resuspend the cell pellet, primarily PMN, in warm (37 &#176;C) RPMI- to a concentration of 107 cells/ml, determined by cell counting using a hemocytometer. Keep the cells at 37 &#176;C until use. Typically, PMN viability and purity are &#62;99% as assessed by trypan blue exclusion and visualization of nuclear morphology after staining, respectively.</li>
</ol>
5. Transuroepithelial Neutrophil Migration Assay
Perform the following steps in a tissue culture hood using aseptic technique. Use only permeable supports bearing confluent 5637 cell layers, as determined in step 2.7. The 24-well plates containing RPMI- can be prepared in advance and kept at 37 &#176;C with 5% CO2 until use.
<ol>
	<li>Aliquot 1 ml warm RPMI- per well in a 24-well plate; prepare 3 wells per permeable support.</li>
	<li>Aspirate the medium from the upper and lower reservoirs of the permeable supports.</li>
	<li>Using sterile forceps, transfer the permeable supports to the 24-well plate prepared in step 5.1. Wash the permeable supports three times by transferring the supports from well to well.</li>
	<li>If a bacterial inoculum is to be used, invert the permeable supports in a sterile 25 mm deep tissue culture dish. If a chemoattractant (e.g., N-Formyl-Met-Leu-Phe (fMLF) or IL-8) is to be used, proceed to step 5.6.</li>
	<li>For experiments with live or killed bacterial stimuli, inoculate the apical side of each permeable support with 60 &#181;l RPMI- (mock infection) or with the bacterial inoculum (6 x 106 CFU) prepared in step 3.5. Place the lid on the dish and incubate at 37 &#176;C with 5% CO2 for 1 hr.</li>
	<li>Add 0.6 ml RPMI- per well to a 24-well low-attachment plate; prepare 1 well per permeable support. Prepare 3 wells containing 0.5 ml RPMI- to enumerate PMN input.
	<ol>
		<li>If a chemoattractant is being used in place of bacteria, add the chemoattractant to 0.6 ml RPMI- in the 24-well plate prepared in step 5.6.</li>
	</ol>
	</li>
	<li>Right the permeable supports into the 24-well low-attachment plate.</li>
	<li>Add 0.1 ml PMN (106 PMN), prepared in step 4.9, to the upper reservoir (basolateral side) of each permeable support. Add 100 &#181;l PMN directly to wells containing 500 &#181;l RPMI-. Incubate at 37 &#176;C with 5% CO2 for 1 hr.</li>
	<li>Using sterile forceps, gently scrape the membrane of the permeable support against the edge of the well to remove additional PMN from the apical side and then dispose of the support.</li>
	<li>Collect PMN by gently scraping the bottom of each well with the 1,000&#160;&#181;l pipette tip, and transfer PMN suspensions to microfuge tubes.</li>
	<li>Enumerate PMN using a hemocytometer.</li>
	<li>To calculate the total number of PMN in the lower reservoir, multiply the number of PMN in 1 mm2 (100 nl) by 6,000. Data can be reported as a percentage of input PMN, or as PMN numbers normalized to 106&#160;input PMN.</li>
</ol>

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	<li>Boyden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemotactic+effect+of+mixtures+of+antibody+and+antigen+on+polymorphonuclear+leucocytes.">The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.</a> J. Exp. Med. 115, 453-466 (1962).</li><li>Zigmond, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orientation+chamber+in+chemotaxis.">Orientation chamber in chemotaxis.</a> Methods Enzymol. 162, 65-72 (1988).</li><li>Zicha, D., Dunn, G. A., Brown, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+direct-viewing+chemotaxis+chamber.">A new direct-viewing chemotaxis chamber.</a> J. Cell Sci. 99 (Pt. 4), 769-775 (1991).</li><li>Chin, A. C., Parkos, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathobiology+of+neutrophil+transepithelial+migration:+implications+in+mediating+epithelial+injury.">Pathobiology of neutrophil transepithelial migration: implications in mediating epithelial injury.</a> Annu. Rev. Pathol. 2, 111-143 (2007).</li><li>Zemans, R. L., Colgan, S. P., Downey, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transepithelial+migration+of+neutrophils:+mechanisms+and+implications+for+acute+lung+injury.">Transepithelial migration of neutrophils: mechanisms and implications for acute lung injury.</a> Am. J. Respir. Cell Mol. Biol. 40, 519-535 (2009).</li><li>Loughman, J. A., Hunstad, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuation+of+human+neutrophil+migration+and+function+by+uropathogenic+bacteria.">Attenuation of human neutrophil migration and function by uropathogenic bacteria.</a> Microbes Infect. 13, 555-565 (2011).</li><li>Lau, M. E., Loughman, J. A., Hunstad, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=YbcL+of+uropathogenic+Escherichia+coli+suppresses+transepithelial+neutrophil+migration.">YbcL of uropathogenic Escherichia coli suppresses transepithelial neutrophil migration.</a> Infect. Immun. 80, 4123-4132 (2012).</li><li>Loughman, J. A., Hunstad, D. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+the+human+leukocyte+enzymes+myeloperoxidase+and+eosinophil+peroxidase.">Assay of the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase.</a> J. Immunol. Methods. 126, 125-133 (1990).</li><li>Lee, W. Y., Chin, A. C., Voss, S., Parkos, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+neutrophil+transepithelial+migration.">In vitro neutrophil transepithelial migration.</a> Methods Mol. 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The authors have nothing to disclose.

Using a cultured bladder epithelial cell line and freshly isolated human PMN, we established an in vitro model of transuroepithelial neutrophil migration. This model has been instrumental in beginning to dissect the complexities of the innate immune response during urinary tract infection (UTI), an extremely common bacterial infection typically caused by UPEC9. During infection of the bladder, or cystitis, recruitment of PMN to the bladder lumen is essential for bacterial clearance10. To es...

O movimento das células através do corpo, muitas vezes através de longas distâncias, é necessário para o crescimento e desenvolvimento, a cicatrização de feridas, e a resposta imune. A migração celular é complexo e requer coordenação de muitos processos diferentes, incluindo cascatas de sinalização e o rearranjo dos componentes do citoesqueleto. As células podem se mover de forma aleatória (quimiocinese), bem como para gradientes químicos definidos (quimiotaxia). Muitas técnicas têm sido desenvolvidas para estudar a migração celular in vitro. A técnica mais antiga e mais comum, a câmara de Boyden, que consiste num sistema de duas câmaras verticais, onde uma substância quimioatractiva é colocada na câmara inferior e as células de interesse são colocados na parte superior da câmara 1. O movimento das células através do filtro permeável, com poros de tamanho definido, que separa as duas câmaras é controlada. Outras técnicas têm sido desenvolvidas para investigar a migração de células, incluindo a câmara de Zigmond 2 e a câmara de Dunn3. Estas abordagens coletivas produziram um panorama significativo para o movimento de muitos tipos celulares diferentes. Além interrogar os princípios básicos da quimiocinese e quimiotaxia, ensaios de duas câmaras têm facilitado a investigação da migração de células através de componentes da matriz extracelular e as duas camadas de células endoteliais e epiteliais. Uma vantagem de sistemas de duas câmaras em relação a outras técnicas é que a membrana porosa pode ser revestida com proteínas tais como colagénio ou fibrinogénio, e a migração de células através de uma barreira de matriz extracelular, como pode ser apreciado. Além disso, as linhas de células cultivadas pode ser cultivada e diferenciado sobre os suportes permeáveis. Para investigar o movimento das células através de uma barreira endotelial, células endoteliais cultivadas são semeadas e cultivadas no reservatório superior dos suportes permeáveis. Células móveis, tais como células do sistema imunológico, são adicionados ao reservatório superior e a migração para dentro do reservatório inferior do outro lado da enbarreira endotelial no sentido fisiológico apical-para-basolateral é observada. Este modelo tem sido inestimável na compreensão de extravasamento de células do sistema imunológico para fora da corrente sanguínea. Em contraste com a migração transendotelial, o movimento das células através de uma barreira epitelial ocorre tipicamente no sentido basolateral-para-apical. A fim de modelar estas eventos in vitro, os investigadores sementes e crescer as células epiteliais cultivadas na parte inferior dos suportes permeáveis. Células móveis são adicionados ao reservatório superior e da migração através da barreira epitelial, que representa a direcção basolateral-para-apical, é monitorado. Tais modelos de migração transepitelial têm contribuído significativamente para a nossa compreensão de respostas inflamatórias na superfície das mucosas, principalmente os de pulmão e intestino 4,5. Em contraste, o tráfico de células imunitárias através do epitélio do tracto urinário tem recebido menos atenção. Para reforçar ainda mais a nossa understanding de respostas imune inata no trato urinário durante a infecção com uropatogênica Escherichia coli (UPEC), desenvolvemos um ensaio in vitro, o ensaio de migração de neutrófilos transuroepithelial, que permite a investigação de leucócitos polimorfonucleares (PMN; neutrófilos) o movimento através de uma barreira epitelial da bexiga 6 -8. Tal como com outros modelos de duas câmaras de migração transepitelial, bexiga células epiteliais humanas cultivadas são crescidas na parte inferior de um suporte permeável e formar camadas epiteliais confluentes. Os neutrófilos humanos, isolados a partir de sangue venoso, são aplicados para o lado basolateral da camada epitelial, e migração através do epitélio na direcção basolateral-para-apical fisiologicamente relevante é quantificada em resposta à infecção por diferentes estirpes de E. coli ou a presença de moléculas de quimioatratantes. Muita investigação focou no movimento de neutrófilos, tanto quimiocinese e quimiotaxia, na ausência de tipos de células adicionais. O ensaio de migração de neutrófilos transuroepithelial é vantajosa, uma vez que leva em conta as interações complexas entre as células epiteliais da bexiga e células do sistema imunológico durante a infecção. Este modelo in vitro tratável tem o potencial de permitir a investigação aprofundada das respostas imunitárias em superfícies uroepiteliais.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Transwell Inserts (i.e.&#160;permeable supports)</td>
			<td>Corning</td>
			<td>3472</td>
			<td>6.5 mm, 3 &#956;m pore, polyester membrane insert</td>
		</tr>
		<tr>
			<td>BD Vacutainer Blood Collection Tubes</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>366480</td>
			<td>16 mm x 100 mm x 10 ml green cap tubes containing sodium heparin</td>
		</tr>
		<tr>
			<td>BD Safety-Lok Blood Collection and Infusion Set</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>367281</td>
			<td>21 G x 0.75 in needle x 12 in tubing</td>
		</tr>
		<tr>
			<td>BD Vacutainer one-use, nonstackable holder</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>364815</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>D4876</td>
			<td>From Leuconostoc mesenteroides</td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS density centrifugation solution</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Low Attachment Plates</td>
			<td>Corning</td>
			<td>3473</td>
			<td>24-well, clear flat bottom</td>
		</tr>
		<tr>
			<td>N-Formyl-Met-Leu-Phe (fMLF)</td>
			<td>Sigma-Aldrich</td>
			<td>F3506</td>
			<td>Reconstituted in DMSO to 10 mM</td>
		</tr>
		<tr>
			<td>Recombinant Human CXCL8/IL-8</td>
			<td>R&#38;D Systems</td>
			<td>208-IL</td>
			<td>Reconstituted in PBS to 100 &#956;g/ml</td>
		</tr>
	</tbody>
</table>

quantitative assessment of human neutrophil migration across a cultured bladder epithelium

O recrutamento de células do sistema imunológico da periferia para o local da inflamação é um passo essencial na resposta imune inata a qualquer superfície mucosa. Durante a infecção da bexiga urinária, os leucócitos polimorfonucleares (PMN; neutrófilos) migram da corrente sanguínea e atravessar o epitélio da bexiga. A incapacidade de resolver a infecção, na ausência de uma resposta neutrofílica demonstra a importância de PMN em defesa da bexiga. Para facilitar a colonização do epitélio da bexiga, uropathogenic Escherichia coli (UPEC), o agente causador da maioria das infecções do tracto urinário (ITU), atenuar a resposta inflamatória aguda, utilizando uma variedade de mecanismos parcialmente definidas. Para investigar ainda mais a interacção entre o hospedeiro eo agente patogénico bacteriano, foi desenvolvido um modelo in vitro do presente aspecto da resposta imune inata a UPEC. No ensaio de migração de neutrófilos transuroepithelial, uma variação na câmara de Boyden, epith bexiga cultivadasElial células são cultivadas até à confluência na parte inferior de um suporte permeável. PMN são isolados a partir de sangue venoso humano e são aplicados para o lado basolateral das camadas de células epiteliais da bexiga. Migração PMN representando a direcção basolateral-para-apical fisiologicamente relevante, em resposta a uma infecção bacteriana ou moléculas quimioatratantes é enumerada utilizando um hemocitómetro. Este modelo pode ser usado para investigar interacções entre UPEC e células eucarióticas, bem como para interrogar os requisitos moleculares para a passagem do epitélio da bexiga por PMN. O modelo de migração de neutrófilos transuroepithelial irá promover nossa compreensão da resposta inflamatória inicial para UPEC na bexiga.

The transuroepithelial neutrophil migration assay enables the quantitative assessment of human PMN migration across cultured bladder epithelial cell layers in response to various stimuli (Figure 1B). While the protocol is straightforward, there are a number of variables that can influence PMN migration and consequently affect the reproducibility of this assay. Measures should be taken while preparing the permeable supports and the PMN to reduce variability between technical and biological replicates. For...

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Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

We developed an in vitro model that mimics an important component of the acute inflammatory response during infection of the bladder with uropathogenic Escherichia coli. The transuroepithelial neutrophil migration assay enables quantitative assessment of human neutrophil migration across bladder epithelia, cultured on permeable supports, in response to bacterial infection or chemoattractant substances.

Avaliação quantitativa de neutrófilos humanos migração através de um Cultivadas bexiga Epitélio

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. ...

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Research

JoVE Journal

Immunology and Infection

14.9K visualizações.  Washington University School of Medicine. We developed an in vitro model that mimics an important component of the acute inflammatory response during infection of the bladder with uropathogenic Escherichia coli. The transuroepithelial neutrophil migration assay enables quantitative assessment of human neutrophil migration across bladder epithelia, cultured on permeable supports, in response to bacterial infection or chemoattractant substances.