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>

<ol>
	<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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+indoleamine+2,3-dioxygenase+by+uropathogenic+bacteria+attenuates+innate+responses+to+epithelial+infection.">Induction of indoleamine 2,3-dioxygenase by uropathogenic bacteria attenuates innate responses to epithelial infection.</a> J. Infect. Dis. 205, 1830-1839 (2012).</li><li>Foxman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+urinary+tract+infection.">The epidemiology of urinary tract infection.</a> Nat. Rev. Urol. 7, 653-660 (1038).</li><li>Haraoka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+recruitment+and+resistance+to+urinary+tract+infection.">Neutrophil recruitment and resistance to urinary tract infection.</a> J. Infect. Dis. 180, 1220-1229 (1999).</li><li>Billips, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+host+innate+immune+response+in+the+bladder+by+uropathogenic+Escherichia+coli.">Modulation of host innate immune response in the bladder by uropathogenic Escherichia coli.</a> Infect. Immun. 75, 5353-5360 (2007).</li><li>Bozeman, P. M., Learn, D. B., Thomas, E. 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. Biol. 341, 205-215 (2006).</li><li>S&#228;ve, S., Mohlin, C., Vumma, R., Persson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+adenosine+A2A+receptors+inhibits+neutrophil+transuroepithelial+migration.">Activation of adenosine A2A receptors inhibits neutrophil transuroepithelial migration.</a> Infect. Immun. 79, 3431-3437 (2011).</li><li>Agace, W. W., Patarroyo, M., Svensson, M., Carlemalm, E., Svanborg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+induces+transuroepithelial+neutrophil+migration+by+an+intercellular+adhesion+molecule-1-dependent+mechanism.">Escherichia coli induces transuroepithelial neutrophil migration by an intercellular adhesion molecule-1-dependent mechanism.</a> Infect. Immun. 63, 4054-4062 (1995).</li><li>Hung, C. S., Dodson, K. W., Hultgren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+urinary+tract+infection.">A murine model of urinary tract infection.</a> Nat. Protoc. 4, 1230-1243 (2009).</li></ol>

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...

The movement of cells throughout the body, often across long distances, is required for growth and development, wound healing, and immune response. Cell migration is complex and requires the coordination of many different processes, including signaling cascades and the rearrangement of cytoskeletal components. Cells can move randomly (chemokinesis) as well as toward defined chemical gradients (chemotaxis). Many techniques have been developed to study cell migration in vitro. The oldest and most common technique, the Boyden chamber, consists of a vertical two-chamber system where a chemoattractant substance is placed in the bottom chamber and cells of interest are placed in the top chamber1. The movement of cells across the permeable filter, with pores of defined size, separating the two chambers is monitored. Additional techniques have been developed to investigate cell migration, including the Zigmond chamber2 and the Dunn chamber3. These collective approaches have yielded significant insight into the movement of many different cell types.
In addition to interrogating the basic principles of chemokinesis and chemotaxis, two-chamber assays have facilitated the investigation of cell migration through extracellular matrix components and both endothelial and epithelial cell layers. An advantage of two-chamber systems over other techniques is that the porous membrane can be coated with proteins such as collagen or fibrinogen, and cell migration across an extracellular matrix-like barrier can be assessed. Additionally, cultured cell lines can be grown and differentiated on the permeable supports. To investigate the movement of cells across an endothelial barrier, cultured endothelial cells are seeded and grown in the upper reservoir of the permeable supports. Motile cells, such as immune cells, are added to the upper reservoir and migration into the lower reservoir across the endothelial barrier in the physiologic apical-to-basolateral direction is observed. This model has been invaluable in understanding extravasation of immune cells out of the blood stream. In contrast to transendothelial migration, the movement of cells across an epithelial barrier typically occurs in the basolateral-to-apical direction. In order to model these events in vitro, researchers seed and grow cultured epithelial cells on the underside of the permeable supports. Motile cells are added to the upper reservoir and migration across the epithelial barrier, representing the basolateral-to-apical direction, is monitored. Such models of transepithelial migration have significantly contributed to our understanding of inflammatory responses at mucosal surfaces, particularly those of the lung and gut4,5.
In contrast, immune cell trafficking through the epithelium of the urinary tract has received much less attention. To further our understanding of innate immune responses in the urinary tract during infection with uropathogenic Escherichia coli (UPEC), we developed an in vitro assay, the transuroepithelial neutrophil migration assay, that enables investigation of polymorphonuclear leukocyte (PMN; neutrophil) movement across a bladder epithelial barrier 6-8. As with other two-chamber models of transepithelial migration, cultured human bladder epithelial cells are grown on the underside of a permeable support and form confluent epithelial layers. Human neutrophils, isolated from venous blood, are applied to the basolateral side of the epithelial layer, and migration across the epithelium in the physiologically relevant basolateral-to-apical direction is quantified in response to infection with different strains of E. coli or the presence of chemoattractant molecules. Much research had focused on neutrophil movement, both chemokinesis and chemotaxis, in the absence of additional cell types. The transuroepithelial neutrophil migration assay is advantageous as it takes into account complex interactions between bladder epithelial cells and immune cells during infection. This tractable in vitro model has the potential to permit the detailed investigation of immune responses at uroepithelial surfaces.

<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

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. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.

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...

Watch this Scientific Journal Video about Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium at JoVE.com

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.

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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Immunology and Infection

14.9K Views.  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.

Video: Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...