Establishment and Characterization of UTI and CAUTI in a Mouse Model

Podsumowanie

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work’s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Streszczenie

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

Wprowadzenie

Urinary tract infections (UTIs) are one of the most common bacterial infections and can be divided into two categories based on the mechanism of acquisition, community and nosocomial acquired UTI. Community-acquired UTIs often occur in otherwise healthy women and studies have shown that approximately 50% of women will have at least one UTI in their lifetime ¹. Additionally, recurrence is a major problem. A woman who has an initial acute infection has a 25-40% chance of having a second infection within six months despite appropriate antibiotic treatment and many women continue to have frequent recurrences ². The bacteria that cause these infections are also becoming increasingly antibiotic resistant further confounding treatment protocols ^3-6. UTI affect millions of individuals every year costing approximately 2.5 billion dollars in health care related expenses in the US, underscoring the impact and prevalence of the disease ^1,7.Nosocomial acquired UTIs are mainly associated with the presence of foreign bodies such as indwelling catheters. Catheter-associated UTIs (CAUTI) remain the most common nosocomial acquired UTI, accounting for ~70-80% of such infections ⁸. Furthermore, CAUTI is associated with increased morbidity and mortality and it is the most common cause of secondary bloodstream infections ⁹.

UPEC associated community acquired UTIs are thought to be caused by the introduction of bacteria into the bladder from reservoirs in the gastrointestinal tract via mechanical manipulation during sexual intercourse, poor hygiene or other microbial population dynamics between different host niches ¹⁰. Once inside the bladder, UPEC employ numerous virulence factors, including capsule, iron acquisition systems, toxins, a virulence plasmid, tRNAs, pathogenicity islands and colonization factors that have been shown to play a role in pathogenesis ^11-14. Critical to the establishment of UPEC colonization, UPEC also encode multiple types of adhesive chaperone usher pathway (CUP) pili that recognize receptors with stereochemical specificity ¹⁵. Type 1 pili, tipped with the FimH adhesin, are expressed by UPEC and bind mannosylated uroplakins ¹⁶ and α-1, β-3 integrins ¹⁷, which are expressed on the luminal surface of both human and mouse bladders ¹⁸. These FimH-mediated interactions facilitate bacterial colonization and invasion of the superficial epithelial cells^19,20. Once inside the cell, UPEC can escape into the cytoplasm where a single bacterium can rapidly divide to form an intracellular bacterial community (IBC), which upon maturation, can contain ~10⁴ bacteria ²¹. IBC formation has been demonstrated in at least six different mouse strains, C3H/HeN, C3H/HeJ, C57Bl/6, CBA, FVB/NJ and BALB/c, and with a wide variety of different UPEC strains and other Enterobacteriaceae ^22-24. However temporal and spatial differences of IBC formation can vary depending upon the mouse background and the infecting UPEC strain. In C3H/HeN mice infected with the prototypical UPEC strains UTI89 or CFT073, IBC formation can be visualized as small biomasses of bacteria as early as 3 hpi (hr post infection). This community continues to expand and reaches a “midpoint” of development approximately 6 hpi when the rod shaped bacteria occupy a large percentage of the cytoplasmic space of terminally differentiated superficial umbrella cells These early IBCs form in a relatively synchronous manner with the majority displaying similar dimensions and morphologies. ~8 hpi the bacteria in the IBC change from a bacilli to cocci morphology. IBCs are transient in nature. Thus, IBC maturation from 12-18 hpi results in continued expansion of the bacterial population, followed by their filamentation and dispersal out of the cell with subsequent spread to neighboring cells ²³. Thus, the IBC niche allows for rapid bacterial growth in an environment protected from host immune responses and antibiotics ²⁵. The distinct stages of UPEC infection that are seen in mice are also observed in humans, such as IBCs and filamentation, supporting the mouse model of UTI as a beneficial tool that can be used to model UTI in humans ^22,26-28.

While a majority of women experience a UTI in their lifetime, the outcome of these infections can range from acute self-limiting infection with no recurrence, to frequent recurrent cystitis. Further, studies have shown a strong familial occurrence of UTI, suggesting a genetic component contributes to UTI susceptibility ²⁹. We have found that the differing UTI outcomes seen in clinics can be mirrored by the differing outcomes of experimental UPEC infection among inbred mouse strains ³⁰. For example, C3H/HeN, CBA, DBA, and C3H/HeOuJ mice are susceptible, in an infectious dose-dependent manner, to long-lasting, chronic cystitis characterized by persistent, high titer bacteria (>10⁴ colony forming units (CFU)/ml), high titer bacterial bladder burdens at sacrifice >4 weeks post-infection (wpi), chronic inflammation, and urothelial necrosis . These mice also display elevated serum levels of IL-6, G-CSF, KC, and IL-5 within the first 24 hpi that serve as biomarkers for the development of chronic cystitis. This may accurately represent the natural course of UTI in some women, as placebo studies have shown that a large percentage of women experiencing UTI will retain high levels of bacteria in their urine for several weeks after the first symptoms of cystitis if not given antibiotic treatment ^31,32. Further, using C3H/HeN mice, we found that a history of chronic cystitis is a significant risk factor for subsequent severe recurrent infections. Recurrent UTI is the most significant clinical manifestation of UTI and the C3H/HeN mouse is currently the only studied model that recapitulates an increased predisposition after previous exposure. A second UTI outcome is recapitulated in C57Bl/6 mice where acute UPEC infection is self-limiting, with resolution of bladder inflammation and bacteriuria within approximately a week. Interestingly, in this model, UPEC readily form quiescent intracellular reservoirs within the bladder tissue from which UPEC are capable of emerging from a dormant state to reinitiate an active UTI, potentially explaining one mechanism for same strain recurrent UTI in humans ^33,34.

In addition to genetic influences on UTI susceptibility, introduction of a catheter into the bladder greatly increases the likelihood of having an infection as well as increasing the range of bacteria able to cause an infection. It has been demonstrated that human urinary catheterization causes histological and immunological changes in the bladder due to mechanical stress that results in a robust inflammatory response, exfoliation, edema of the lamina propria and submucusa, urothelial thinning, and mucosal lesion of the urothelium and kidney ^35,36. Additionally, the catheter provides a surface for bacterial attachment thereby creating an environment utilized by several species to cause CAUTI. While UPEC are still a major contributor, Enterococcus faecalis accounts for 15% of these CAUTI ³⁷. E. faecalis is becoming increasingly resistant to antibiotics with vancomycin resistance emergence, posing a serious health concern ³⁸. E. faecalis possess numerous virulence factors including toxins and adhesins necessary for attachment to both the catheter and epithelium ³⁸. During urinary catheterization, the host is vulnerable to microbial adhesion, multiplication and dissemination in the urinary tract ^39,40. E. faecalis forms a biofilm on the catheter as part of a mechanism to persist in the bladder and disseminate to the kidneys, which is reproduced in a mouse CAUTI model⁴¹. Recently, it has been shown during urinary catheterization, fibrinogen (Fg) is released into the bladder as part of the inflammatory response. Fg accumulates in the bladder, coats the catheter and is essential for E. faecalis biofilm formation, functioning as an attachment scaffold. In a C57BL/6 mouse model of CAUTI, we discovered that E. faecalis biofilm formation on the catheter, and thus persistence in the bladder, was dependent on the Ebp pilus, specifically its tip adhesin EbpA. We found that the N-terminal domain of EbpA specifically binds to Fg coating the catheter. Additionally, it was found that E. faecalis utilizes Fg as metabolite source during infection, thus enhancing biofilm formation ⁴².

Mouse models have proven critical to understanding as well as predicting clinical manifestations of UTI and CAUTI ⁴¹. In this article we demonstrate inoculum preparation of the cystitis UPEC isolate UTI89 and transurethral inoculation of C3H/HeN mice. Additionally, we demonstrate a protocol for catheter insertion in C57BL/6 mice and inoculation of the E. faecalis OG1RF strain. Both of these techniques lead to consistent and reliable UTI or CAUTI in mice. We also display techniques used to observe IBC formation during acute cystitis and urine collection for the analysis of chronic or recurrent cystitis. C3H/HeN mice have been used to study aspects of UPEC pathogenesis including initial bacterial invasion, IBC formation, filamentation and the development of chronic cystitis ^23,33,43. These virulence parameters have also been studied in a variety of other mouse backgrounds ^22,33. For CAUTI, the C57BL/6 model allows for foreign body implantation into the bladder with subsequent bacterial colonization, which can be maintained for 7 days post infection ⁴¹. These models have been useful for assessing bacterial virulence mechanisms, host responses to UTI and mechanisms to subvert host responses, much of which has been subsequently recapitulated or observed in clinical human populations.

Protokół

Ethics statement: The Washington University Animal Studies Committee approved all mouse infections and procedures as part of protocol number 20150226, which expires 12/10/2018. Overall care of the animals was consistent with The Guide for the Care and Use of Laboratory Animals from the National Research Council and the USDA Animal Care Resource Guide. Euthanasia procedures are consistent with the “AVMA guidelines for the Euthanasia of Animals 2013 edition.”

1. UPEC UTI Protocol, Inoculation Needle Preparation (Figure S1)

1. Remove the cap of the 30 G needle. Thread approximately 1 inch of PE10 tubing onto the shaft of the needle. UV sterilize the needle assemblies overnight. Catheterized needles can be stored indefinitely in sterile petri plates.

2. UPEC Bacterial Inoculum Preparation

1. Prepare UTI89 inoculum (start 72 hr prior to the inoculation day)
   1. Streak UTI89 onto an LB (Luria-Bertani) agar plate from a frozen stock. Incubate overnight at 37 °C. Pick a colony and inoculate it into 10 ml of LB in a 125 ml flask. Incubate at 37 °C for 24 hr, statically.
   2. Inoculate 10 µl of overnight culture into 10 ml of LB in a new 125 ml flask for an additional 24 hr at 37 °C, statically.
   3. Transfer 3 ml (will vary based on strain and desired inoculum size) to sterile 1.5 ml tubes and centrifuge at 7,000 x g for 3 min and resuspend the pellet in 1x PBS and centrifuge a second time. Resuspend the bacterial pellet in 1 ml 1x PBS.
2. Measure bacterial optical density and adjust concentration to the desired inoculum density. The standard concentration of the inoculum used is 10⁷ bacteria; however, this may vary due to the design of the study.

3. Bacterial Inoculation

1. Clean the workstation with 70% ethanol and cover area with absorbent paper (or use sterile flow hood).
2. Draw up to 0.9 ml of the prepared bacterial inoculum into a 1 ml (TB) syringe (remove air bubbles). Attach a prepared sterile inoculation needle with PE10 tubing, onto the syringe containing the inoculum, then sterilely trim the polyethylene tubing.
   NOTE: Leave 1 mm of tubing above the tip of the needle to avoid puncturing the bladder.
3. Cut a 1 inch square piece of parafilm and put a dab of surgical lubricant (approximately the size of a dime) on top.
4. Anesthetize female C3H/HeN mice by putting them in a vaporizer chamber (following manufactures protocol) until unconscious but still breathing normally (1 breath/sec).
   NOTE: Some IACUC committees do not approve the use of a vaporizer. Please follow the indication of the IACUC committee of your institution.
   CAUTION: Isoflurane is an inhalation anesthetic. Use in a well-ventilated area and minimize inhalation.
5. Remove the mouse from the vaporizer and place it on its back on a paper towel and spread the legs.
6. Cover the nose of the mouse with a nose cone (a tube connected to the vaporizer that provides a controlled isoflurance dose that comes equipped on some vaporizer units) to maintain anesthetization.
7. Gently palpitate the bladder to induce urination and ensure a voided bladder. Wipe the periurethral area with 100% ethanol wipe. Dab the inoculation needle/syringe, point first, into the surgical lubricant.
8. Inoculate each mouse with 50 µl of the bacteria solution by inserting the inoculation needle transurethrally, approximately 12 mm, and pressing down on the syringe plunger gently to dispense the inoculum into the bladder gently (10 µl/sec). Remove the inoculation needle from the mouse.
   NOTE: Immediate return of inoculum at the urethral opening when beginning to inoculate indicates improper or incomplete insertion of the needle.
9. Remove the mouse from nose cone and return it to its cage. Repeat steps 3.3-3.8 for each mouse. When/if switching inoculum conditions/strains, dispose of the syringe and inoculation needle in an approved sharps container and start again step 3.2.  
   NOTE: Inoculation with uropathogenic organisms generally does not cause severe pain symptoms. However, in rare instances, administering large doses of pathogens may cause fever, reduction in food and water intake and abnormal behavior. Animal health should be monitored throughout the experiment. If overt pain symptoms are notice, an analgesic, such as Buprenorphine (0.05−0.1 mg/kg given subcutaneously), can be applied. Procedures should be in accordance with each institution’s IACUC.

4. Determination of Bacterial Burdens

1. Prepare separate 5 ml tubes for each bladder and kidneys pair to be harvested. Add 1ml of 1x PBS/tube/mouse bladder to be harvested. Add 0.8 ml of 1x PBS/tube/pair of mouse kidneys to be harvested. Label each tube to correspond to a given mouse. Prepare a 96-well plate containing 180 µl of 1x PBS in each well for 1:10 serial dilutions.
2. Clean the work area with 70% ethanol and cover the area with an absorbent cover or paper towel.
3. Anesthetize the mouse with isoflurane (see step 3.4) until the mouse stops breathing for approximately 1 min, then place it on the absorbent cover or paper towel. Other methods of euthanasia are also accepted by IACUC committees, such as carbon dioxide, and can be substituted for isoflurane overdose.
4. Sacrifice the mouse by rapid cervical dislocation that consists of restraining the neck at the base of the head and pulling the tail horizontally away from the body until dislocation occurs.
   NOTE: Most IACUC committees require a secondary means of euthanasia and cervical dislocation is a common method. However, other methods can be used if approved by the IACUC committee.
5. Place the dead mouse on its back and spray 70% ethanol on the abdomen. Dissect the mouse, harvest the bladder and kidneys, in that order to minimize potential contamination from the blood following the kidney dissection. Place the organs independently into the prepared 5 ml tubes containing 1x PBS (see step 4.1).
   NOTE: Use different scissors for the external dissection and for organ harvesting to minimize contamination.
6. Tap the tube to ensure the organs are in the 1x PBS. Repeat step 4.3-4.5 for each mouse. Clean scissors and forceps in 70% ethanol after each mouse.

5. Bacterial Recovery

1. Homogenize harvested bladder and kidneys in the 5 ml tubes with a tissue homogenizer, 15 sec per kidney and 30 sec per bladder. Rinse homogenizer sequentially in 1x PBS, 70% ethanol and 1x PBS between samples.
2. Make serial 1:10 dilutions out to 10^-8 and plate each dilution for CFU (colony forming units) on LB agar plates. Incubate the plates at 37 °C overnight and count colonies the next day.

6. IBC Enumeration

1. Aseptically remove the bladder and place it in 1x PBS in a 6-well plate with a silicone bottom. Plates can be prepared using a silicone elastomer kit and pouring approximately 1 cm of silicone in each well, see manufactures instructions. Cut the bladder in half using scissors.
2. Using small metal pins gently splay the bladder so that the bladder lumen is facing upward. Be sure to place the pins at the outermost edges of the bladder sections to ensure the maximal amount of urothelium is exposed.
3. After splaying, gently wash the bladder once with 1x PBS. Remove the 1x PBS by pipet. Fix the bladder by adding enough 3% paraformaldehyde to cover the entire splayed bladder. CAUTION: Paraformaldehyde is carcinogenic. Proper protective equipment, including gloves, should be worn at all times. Disposal should follow institutional guidelines.
4. Incubate at room temperature for 1 hr. Remove paraformaldehyde solution. Wash once with 1x PBS.
5. Wash with LacZ wash solution (2 mM MgCl₂, 0.01% sodium deoxycholate and 0.02% nonidet-p40in 1x PBS) three times for 5 min per wash.
6. Remove wash solution and add enough LacZ stain (9.5 ml LacZ wash solution, 0.4 ml 25 mg/ml X-gal and 0.1 ml of 100 mM K-ferrOcyanide/K-ferrIcyanide) to cover the bladders. Incubate at 30 °C overnight protected from light.
7. Remove splayed bladders from incubator and observe IBCs, which appear as blue puncta as visualized with a dissecting scope, 40-60X magnification.
   NOTE: Sometimes a mouse may urinate prior to placement of the tube under the urethra. In this case, wait 15-20 min before attempting to collect urine again.

7. Urine Collection for Bacteriuria CFU Enumeration (Not Applicable for CAUTI)

1. To collect urine pre or post infection gently restrain the mouse by holding the tail and placing it on a flat elevated surface (top of mouse cage).
2. Hold a sterile 1.5 ml tube under the mouse urethra. Gently press down on the back of the mouse near the tail to apply pressure to the bladder. Catch the urine in the 1.5 ml tube.
3. Make serial 1:10 dilutions out to 10^-7 and plate each dilution on appropriate LB. Incubate the plates at 37 °C overnight and count colonies the next day.

8. CAUTI Model Protocol, Catheter Needle Preparation for CAUTI Model (Figure S2)

1. Remove the cap of the 30 G needle. Cut a 7 mm piece of PE10 tubing and 5 mm piece of silicone tubing such as RenaSIL. Thread the needle with PE10 tubing until the tubing touches the base of the needle.
2. Then feed the 5 mm piece onto the PE10-containing needle. UV sterilize the needles assemblies overnight.
   NOTE: When loading the silicone tubing, leave approximately 1 mm of the tubing extending past the needle tip.

9. E. faecalis OG1RF Bacterial Inoculum Preparation

1. Prepare OG1RF inoculum starting 48 hr prior to the inoculation day. Streak OG1RF onto a BHI (brain heart infusion) plate from a frozen stock.
2. Pick a colony and inoculate it into 10 ml of BHI and grow it statically for 18 hr at 37 °C. Centrifuge the culture and wash the pellet (3 times) in 1 ml volumes of sterile 1x PBS.
3. Resuspend the bacterial pellet into 1x PBS. Measure bacterial optical density and adjust the concentration to the desired inoculum size. The standard concentration of the inoculum used is 10⁷; however, this may vary due to the design of the study

10. Catheter Implantation

1. Clean the workstation with 70% ethanol and cover the area with an absorbent cover (or use sterile flow hood).
2. Attach catheter-needle to an empty 1 ml (TB) syringe. Cut a 1 inch square piece of parafilm and put a dab of surgical lubricant (approximately the size of a dime) on top.
   NOTE: Two different needles are used for deposition of the catheter and for the inoculum to minimized accidental inoculation due to the mechanical manipulation necessary for catheter implantation. This also allows for the convenience of preparing fewer inoculation needles.
3. Anesthetize C57BL/6 mice by putting them in a 32 ounce glass jar containing a tea-infuser ball with cotton balls soaked in 3 ml of isoflurane or vaporizer chamber (following manufactures protocol) until unconscious but still breathing normally (1 breath/sec).
4. Then put the mouse on its back on a paper towel and spread the legs. Cover the nose of the mouse with a nose cone or 50 ml conical tube with cotton balls containing a small amount (approximately 1-2 ml) of isoflurane.
   CAUTION: Isoflurane is an inhalation anesthetic. Use in a well-ventilated area and minimize inhalation by researcher.
5. Gently palpitate the bladder to induce urination and ensure a voided bladder. Wipe periurethral area with a 100% ethanol wipe.
6. Disinfect the periurethral area with 10% povidone-iodine solution using a cotton-tip applicator. Dab the inoculation needle/syringe, point first, into the surgical lubricant.
   NOTE: Povidine-iodine is used for catheter implantation, which is a stronger aseptic solution than alcohol. Catheter implantation requires more mechanical manipulation to insert the catheter and thus a greater potential for contamination.
7. Insert the catheter into the urethral opening. Once in, use tweezers to push the (7 mm PE10) long piece toward the bladder, consequently pushing the (5 mm silicone) small catheter and depositing it into the bladder. Remove the needle, with the 7 mm tubing still attached, immediately.

11. Bacterial Inoculation

1. Follow the bacterial inoculation protocol in section 3, using the prepared inoculum from section 9. Remove the mouse from the nose cone and return it to its cage

12. At Each Time Point of Sacrifice

1. Prepare 5 ml tubes for bladder and kidney (For organs follow step 4.1) and 1.5 ml Eppendorf tube for catheters. Add 1 ml of 1x PBS into 1.5 ml Eppendorf tube for catheter samples
2. Follow steps 4.3 to 4.5. Dissect the mouse, harvesting the bladder, kidneys and catheter, placing the tissues independently into 5 ml tubes containing 1x PBS and the catheter into the 1.5 ml Eppendorf (see step 12.1).
   NOTE: Use different scissors for the external dissection and for organ harvesting and catheter retrieval to minimize contamination
3. Then follow step 4.6.

13. Bacterial Recovery

1. For organs, follow steps 5.1 and 5.2. For bacterial recovery from catheters, vortex at maximum speed for 30 sec, sonicate the catheter samples for 5 min using a bath sonicator, and then vortex at maximum speed for an additional 30 sec.
2. Make serial 1:10 dilutions out to 10^-8 and plate each dilution for CFU enumeration on BHI plates. Incubate the plates at 37 ºC overnight and count colonies the next day.

Wyniki

The intravesical models of uncomplicated and catheter associated UTI provide flexible platforms for elucidating the molecular mechanisms of bacterial pathogenesis, the impact of these diseases on host tissue, and the development and testing of novel approaches to manage these common and costly infections. Depending on the mouse strain and pathogen, intravesical inoculation can be used to study host-pathogen interactions to elucidate factors necessary for initiating or modulating acute (Figure 1 and

Dyskusje

Uncomplicated community acquired UTI is a common and costly infection accounting for several million primary care visits every year ⁴⁶. In addition, CAUTIs are a common healthcare acquired infection that has become extremely costly to healthcare providers as the Centers of Medicare and Medicaid Services no longer reimburses providers for the added cost of treatment resulting from hospital acquired CAUTI ⁴⁵. The mouse models of UTI, both uncomplicated cystitis and CAUTI, described in these protocols ...

Materiały

Name	Company	Catalog Number	Comments
Material for catheter and needle preparation:
30 G needles	BD Precision Glide	305106	30 G x ½ (0.3 mm x 13 mm)
PE10 polyethylene tubing	BD	427400	Inside diameter -0.011 in (0.28 mm); outside diameter – 0.024 in (0.61 mm)
RenaSIL 025 platinum cured silicon tubing	Braintree Scientific, Inc	SIL 025	inside diameter-0.012 x outside diameter 0.025, 25 ft coil
Material for infections:
Isoflourane – Isothesia	Butler Schein	29405	250 ml
Clear Glass Straight-Sided Jar	Kimble Chase	5413289V 21
Stainless Steel Tea Infuser	Schefs-Amazon		Premium Loose Leaf Tea Infuser By Schefs - Stainless Steel - Large Capacity -
Non-sterile cotton balls	Fisherbrand	22-456-880
50 ml Falcon tubes	VWR	89039-660
Isotec 3 -vaporizer	Ohmeda	1224478
Ear punch	Fisher Scientific	13-812-201	(when necessary)
Betadine solution	Betadine solution		10% Povidie-iodine topical solution
Q-tips	Fisher Scientific	22-037-924	6 inches
Diapers for bench	Fisherbrand	14206 63	Absorbent Underpads (20”X36”mats)
Surgical lubricant	Surgilube	0281-0205-36
Dissecting scissor	Fine Science tools, INC	14084-08
Micro-Adson Forceps	Fine Science tools, INC	11018-12
1 ml syringe	BD	309659	Tuberculin slip tip
Parafilm	Bemis	PM996	4 inches x 125 FT
Eppendorf rack	Fisherbrand	05-541-1
Eppendorf tubes	MIDSCI	AVX-T-17-C
Harvesting catheters, bladders and kidneys:
Homogenizer	PRO Scientific INC	Bio-Gen Pro 200
5 ml polypropylene round-bottom tube	BD	352063	for organ homogenization
Paper towel	Georgia-Pacific
Ethanol	Pharmco-AAPER	11100020S	200 proof
Costar™ Clear Polystyrene 96-Well Plates	Corning	3788
1x Phosphate sodium saline	Sigma-Aldrich	P3813
BRANSONIC Ultrasonic cleaner 1210	Branson Ultrasonics Corporation	1210
IBC materials:
6-well tissue culture test plate	Techno Plastic Products	92006
Pins	Fine Science Tools	26002-20
Sylgard 184	Dow Corning	3097358-1004	Silicone Elastomer Kit
X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactoside)	Invitrogen	15520-034	Ultrapure
N, N-Dimethylformamide	Sigma Aldrich	D4551
MgCl2 (Magnesium chloride)	Sigma Aldrich	M8266
Sodium deoxycholate	Sigma Aldrich	D6750
Nonidet-P40	Roche	11754599001	Octylphenolpoly(ethyleneglycolether)n
Potassium hexacyanoferrate(II) trihydrate (K-ferrOcyanide)	Sigma Aldrich	P3289
Potassium hexacyanoferrate(III) (K-ferrIcyanide)	Sigma Aldrich	60299