Name: transurethral induction of mouse urinary tract infection
Uploaded: 2010-08-05T17:30:00
Description: Watch this Scientific Journal Video about Transurethral Induction of Mouse Urinary Tract Infection at JoVE.com

Gli autori desiderano ringraziare Shelly Xie e Jennifer Payne per la loro assistenza tecnico esperto. Questo lavoro è stato reso possibile attraverso sovvenzioni da parte della Società di Urologia Pediatrica e lo Stanford Research Pediatric Fondo.

I. Preparazione dei cateteri uretrali <ol><li> Per ogni vescica del mouse e reni da campionare, posto 5-6 grani in acciaio inox (vescica, 1.6 mm o dimensione 0,9-2,0 miscela mm) o perle di ossido di zirconio (rene, 0,5 mm di diametro) in una provetta provetta Microbank ™, rispettivamente. Autoclave il tallone contenenti tubi. </li><li> Dopo i tubi raffreddare a temperatura ambiente, aggiungere 200 microlitri di PBS sterile ad ogni acciaio inox tallone contenenti tubi e 400 microlitri di PBS sterile ad ogni ossido di...

<ol>
	<li>Schaeffer, A. J., Schwan, W. R., Hultgren, S. J., Duncan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+type+1+pilus+expression+in+Escherichia+coli+to+ascending+urinary+tract+infections+in+mice.">Relationship of type 1 pilus expression in Escherichia coli to ascending urinary tract infections in mice.</a> Infect Immun. 55, 373-380 (1987).</li><li>Rouschop, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urothelial+CD44+facilitates+Escherichia+coli+infection+of+the+murine+urinary+tract.">Urothelial CD44 facilitates Escherichia coli infection of the murine urinary tract.</a> J Immunol. 177, 7225-7232 (2006).</li><li>Malaviya, R., Ikeda, T., Abraham, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+mast+cells+to+bacterial+clearance+and+their+proliferation+during+experimental+cystitis+induced+by+type+1+fimbriated+E.+coli.">Contribution of mast cells to bacterial clearance and their proliferation during experimental cystitis induced by type 1 fimbriated E. coli.</a> Immunol LettM. 91, 103-111 (2004).</li><li>Lane, M. C., Alteri, C. J., Smith, S. N., Mobley, H. 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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=In+vivo+dynamics+of+type+1+fimbria+regulation+in+uropathogenic+Escherichia+coli+during+experimental+urinary+tract+infection.">In vivo dynamics of type 1 fimbria regulation in uropathogenic Escherichia coli during experimental urinary tract infection.</a> Infect Immun. 69, 2838-2846 (2001).</li><li>Bishop, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP-regulated+exocytosis+of+Escherichia+coli+from+infected+bladder+epithelial+cells.">Cyclic AMP-regulated exocytosis of Escherichia coli from infected bladder epithelial cells.</a> Nat Med. 13, 625-630 (2007).</li><li>Thumbikat, P., Waltenbaugh, C., Schaeffer, A. J., Klumpp, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen-specific+responses+accelerate+bacterial+clearance+in+the+bladder.">Antigen-specific responses accelerate bacterial clearance in the bladder.</a> J Immunol. 176, 3080-3086 (2006).</li><li>Hagberg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascending,+unobstructed+urinary+tract+infection+in+mice+caused+by+pyelonephritogenic+Escherichia+coli+of+human+origin.">Ascending, unobstructed urinary tract infection in mice caused by pyelonephritogenic Escherichia coli of human origin.</a> Infect Immun. 40, 273-283 (1983).</li><li>Hopkins, W. J., Hall, J. A., Conway, B. P., Uehling, D. 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Induzione di infezioni del tratto urinario del mouse tramite l&#39;inoculazione di batteri transuretrale è una procedura ormai consolidata, ma tecnicamente impegnativo 11. Hung e Hultgren recentemente pubblicato un eccellente rassegna di tecniche transuretrale di infezione del tratto urinario del mouse 13. In questo lavoro, cerchiamo di illustrare ulteriormente i punti più fini di questo approccio sperimentale. I novizi possono praticare la loro tecnica eseguendo una ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cryovial tubes, 2.0 ml polypropylene (no caps)</td>
<td>&nbsp;</td>
<td>E&amp;K Scientific</td>
<td>640201</td>
<td>&nbsp;</td>
<tr>
<td>Caps for cryovial tubes, with 'O' ring</td>
<td>&nbsp;</td>
<td>E&amp;K Scientific</td>
<td>440011-N</td>
<td>&nbsp;</td>
<tr>
<td>Bullet Blender</td>
<td>&nbsp;</td>
<td> Next Advance</td>
<td>BBX24</td>
<td>&nbsp;</td>
<tr>
<td>Zirconium Oxide Beads (0.5mm)</td>
<td>&nbsp;</td>
<td>Next Advance</td>
<td>ZROB05</td>
<td>&nbsp;</td>
<tr>
<td>Stainless Steel Beads (1.6mm)</td>
<td>&nbsp;</td>
<td>Next Advance</td>
<td>SSB16</td>
<td>&nbsp;</td>
<tr>
<td>Stainless Steel Bead Blend (0.9-2.0mm)</td>
<td>&nbsp;</td>
<td>Next Advance</td>
<td>SSB14B</td>
<td>&nbsp;</td>
<tr>
<td>CBA/J Mice, 8-12 week old female</td>
<td>&nbsp;</td>
<td>Jackson Laboratories</td>
<td>000656</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline (NaCl 137mM, KCl 2.7mM, Na2HPO4 4.3mM, KH2PO4 1.4mM)</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>EM-SX0420-13 (NaCl) EM-PX1405-1 (KCl) EM1.06585.0 (Na2HPO4) EM-5108-1 (KH2PO4)</td>
<td>&nbsp;</td>
<tr>
<td>LB agar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LB broth</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>1.10285.0500</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane (Aerrane)</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>NDC 10019-773-60</td>
<td>&nbsp;</td>
<tr>
<td>Inoculating loops</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>22-363-601</td>
<td>&nbsp;</td>
<tr>
<td>Eosin-methylene blue (EMB) agar powder</td>
<td>&nbsp;</td>
<td>Himedia Laboratories</td>
<td>MM022-500G</td>
<td>&nbsp;</td>
<tr>
<td>MacConkey agar powder</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>212123</td>
<td>&nbsp;</td>
<tr>
<td>LB agar powder</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>244520</td>
<td>&nbsp;</td>
<tr>
<td>Intramedic non-radiopaque polyethylene tubing, PE10 [inner dimension 0.28 mm (0.011 inch); outer dimension 0.61 mm (0.024 inch)]</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>427401</td>
<td>&nbsp;</td>
<tr>
<td>30 gauge needles, 1/2 inch</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>305106</td>
<td>&nbsp;</td>
<tr>
<td>1 ml tuberculin Slip-Tip syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>309602</td>
<td>&nbsp;</td>
<tr>
<td>3 ml Luer-Lock syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>309585</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

transurethral induction of mouse urinary tract infection

Uropatogeni ceppi batterici di interesse vengono coltivate su agar. In generale, uropatogeni<em&gt; E. coli</em&gt; (UPEC) e altri ceppi possono essere coltivate pernottamento a Luria-Bertani (LB) agar a 37 ° C nell&#39;aria ambiente. Ceppi UPEC crescere come colonie traslucido bianco-giallastro su agar LB. In seguito alla conferma della morfologia delle colonie del caso, singole colonie vengono poi raccolte per essere coltivate in brodo. LB brodo può essere utilizzato per i ceppi batterici più uropatogeni. Due porte seriali, pernottamento culture brodo LB può essere impiegato per migliorare l&#39;espressione di tipo I pili, un ben definito fattore di virulenza dei batteri uropatogeni. Culture brodo sono diluiti alla concentrazione desiderata in tampone fosfato (PBS). Da otto a 12 settimane i topi vecchi femminile sono posti sotto anestesia isoflurano e transurethrally inoculato con batteri utilizzando tubi di polietilene coperto di siringhe 30 gauge. Inoculi tipico, che deve essere empiricamente determinato per ogni combinazione ceppo batterico / mouse, sono 10<sup&gt; 6</sup&gt; A 10<sup&gt; 8</sup&gt; Ufc per mouse in 10-50 microlitri di PBS. Dopo il periodo di infezione desiderato (un giorno a diverse settimane), i campioni di urina e la vescica ed entrambi i reni vengono raccolte. Ogni organo è tritato, posto in PBS, e omogeneizzato in un omogeneizzatore proiettile blu. Omogenati di urina e di tessuto sono serialmente diluiti in PBS e coltivate su agar appropriato. Il giorno successivo, unità formanti colonie sono contate.

Watch this Scientific Journal Video about Transurethral Induction of Mouse Urinary Tract Infection at JoVE.com

Transurethral Induction of Mouse Urinary Tract Infection

Questo video dimostra metodi per indurre transurethrally infezioni del tratto urinario del mouse e quantificare l&#39;entità delle infezioni che ne derivano.

Induzione transuretrale di infezioni del tratto urinario mouse

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