Materials and Reagents

1. Microcentrifuge tubes (1.5 ml for urine collection and 2.0 ml for tissue homogenization)
   
2. 1 ml syringes (BD, catalog number: 309628 )
   
3. 27 G needles (BD, catalog number: 305136 )
   
4. ELISA plates (Nunc MaxiSorp^® flat-bottom 96 well plate) (Sigma-Aldrich, catalog number: M9410 )
   
5. Unfrosted or frosted glass slides (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10149870 )
   
6. poly-L-lysine-coated glass slides (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: P4981 )
   
7. Coverslips 60 x 24 mm
8. Mice
   Only female mice were used for the UTI infection model because the urethra in male mice is extremely difficult to catheterize due to its anatomy. The following strain of mice at an age of 8 to 16 weeks was used:
   C57BL/6
   The following knock-out mice were successfully used in the model to study the relevance of a number of host-defense genes but these mice are optional and not required to generate the model:
   Tlr4^−/−
   Myd88^−/−
   Trif^−/−
   Trif^Lps2/Lps2
   Il-1β^−/−
   Irf3^−/−
   Note: All knock-out mice were at least 8x backcrossed to C57BL/6 mice. All mice were bred and housed in specific pathogen free conditions. Mice were only used upon permission of the local animal experimental ethics committee.
   
9. Bacteria
   
   1. UPEC strain CFT073, provided by ATCC (ATCC, catalog number: 700928 )
      
   2. tcpC-deficient CFT073 tcpC::kan, generated in the lab using the method described by Datsenko/Wanner (Datsenko and Wanner, 2000).
      
   3. Complemented mutant strain CFT073 tcpC::kan+pTcpC, the plasmid was generated as described (Cirl et al., 2008).
   Note: Bacterial strains used to inoculate the animals were maintained on LB agar plates containing appropriate antibiotics. The strains were grown overnight on tryptic soy agar. Bacteria were harvested in phosphate-buffered saline and bacterial cell density (OD₅₉₇ = 1 corresponded to 1 x 10⁹ bacteria/ml) was subsequently adjusted on the basis of a standard curve of absorbance at 597 nm to a concentration of 10¹⁰ bacteria per ml by dilution in phosphate-buffered saline.
   
10. Ice
    
11. Dry ice
    
12. Ethanol (70%)
    
13. Isoflurane (Abbott Laboratories, Forene^®, catalog number: 506949 )
    
14. Ketamine (Ketaminol^®, catalog number: 511519 )
    
15. Xylazine (Rompun^®, catalog number: 0 22545 )
    
16. 0.9% NaCl
    
17. Phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: D8662 )
    
18. Gentamicin
    
19. Tissue-Tek^® O.C.T. compound (SAKURA FINETEK, catalog number: 4583 )
    
20. Tissue-Tek^® Cryomold (SAKURA FINETEK, catalog number: 4565 )
    
21. Isopentane (Sigma-Aldrich, catalog number: PHR1661 )
    
22. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
    
23. Normal goat serum (Agilent Technologies, catalog number: X0907 )
    
24. Rat monoclonal antibody NIMP-R14 (1: 200) (Abcam, catalog number: ab2557 )
25. Antiserum to a synthetic peptide within the PapG adhesin (1:200), produced in the laboratory
    
26. Goat anti-rat immunoglobulins (1:200), conjugated with Alexa 488 dye (A488; 495ext/519em nm) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: A-11006 )
    
27. Goat anti-rabbit immunoglobulins (1:200), conjugated with Alexa 568 dye (A568; 578ext/603em nm) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: A-11011 )
    
28. Anti-NLRP3 antibody (Santa Cruz Biotechnology, catalog number: sc-66846 )
    
29. Anti-IL-1β antibody (Abcam, catalog number: ab9722 )
    
30. Anti-ASC antibody (Abcam, catalog number: ab155970 )
    
31. DAPI (Sigma-Aldrich, catalog number: D9542 )
    
32. VECTASHIELD mounting medium (Vector Laboratories, catalog number: H-1000 )
    
33. MIP-2 quantification kit (R&D Systems, catalog number: MM200 )
    
34. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
    
35. Tryptic soy agar (TSA) (BD, Difco^TM, catalog number: 236950 )
    
36. LB agar (BD, catalog number: 244520 )
    
37. Columbia 5% blood agar (BD, catalog number: 221263 )
    
38. Sucrose (Sigma-Aldrich, catalog number: 84100 )
    
39. Ketamine/Xylazine solution (see Recipes)
    
40. Media for cultivation of bacteria (see Recipes)
    Tryptic soy agar plates, LB, Columbia 5% blood agar
    
41. Sucrose solution (see Recipes)
    
42. 4% paraformaldehyde (see Recipes)
    

Equipment

1. Burker chamber
   
2. ELISA washer
   
3. Drop glass jar for gas anesthesia
   
4. Biosafety hood in biosafety level 2 facility
   
5. Curved forceps, scissors
   
6. Soft polyethylene catheter (0.61 mm outer diameter; Clay Adams) (BD, catalog number: 427401 )
   
7. Stomacher 80 homogenizer (Seward medical, catalog number: 0080/000/EU )
   
8. Cryostat sections were made with a Microtome blade C-35 (FEATHER Safety Razor, model: C-35)
   
9. Fluorescence microscopy (Olympus, model: AX60 , equipped with filter sets [excitation/emission] 495ext/519em nm and 578ext/603em nm)
   
10. AxioCam ERc 5s camera (Zeiss, model: AxioCam ERc 5s)
    
11. Labsystems Multiskan PLUS reader (Analytical Instruments, Golden Valley, USA)
    

Procedure