Materials and Reagents

1. Cell lines: INT 407, Hep-2, Hela, HT-29 (available from ATCC and other commercial sources)
   
2. Vibrio cholerae classical biotype strain O395
   
3. Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Gibco^®)
   1. High glucose (4,500 mg/L) (catalog number: 12800-017 )
      
   2. Low glucose (1,000 mg/L) (catalog number: 31600-034 )
      
   3. No glucose (catalog number: 11966-025 )
      
4. New born calf serum (NCS) (not heat inactivated) (Life Technologies, Gibco^®, catalog number: 16010-159 )
   
5. 10x Trypsin-EDTA (Life Technologies, Gibco^®, catalog number: 15400 )
   
6. TritonX-100 (Affymetrix, catalog number: T1001 )
   
7. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: C6164 )
   
8. D-(+)-Glucose (Sigma-Aldrich, catalog number: G7021 )
   
9. D-Galactose
   
10. 10x cell lysis buffer (Cell Signaling Technology, catalog number: 9803 )
    
11. pNPP (5 mg) (Sigma Chemical, catalog number: N-9389 )
    
12. Bacto tryptone (BD, Difco^TM, catalog number: 211705 )
    
13. Bacto yeast extract (BD, Difco^TM, catalog number: 212750 )
    
14. Bacto agar (BD, Difco^TM, catalog number: 214010 )
    
15. NaHCO₃ (Merck KGaA)
    
16. NaCl (Merck KGaA)
    
17. Na₂HPO₄ (Merck KGaA)
    
18. KCl (Merck KGaA)
    
19. KH₂PO₄ (Merck KGaA)
    
20. 70% ethanol (Merck KGaA)
    
21. NaOH (Merck KGaA)
    
22. Personal protective items (apron, gloves, mask etc.)
    
23. Bio-Rad protein assay dye reagent (Bio-Rad Laboratories)
    
24. Complete DMEM (100 ml) (see Recipes)
    
25. Incomplete DMEM (100 ml) (see Recipes)
    
26. No glucose complete DMEM (100ml) (see Recipes)
    
27. 10x PBS (500 ml, pH 7.4) (see Recipes)
    
28. 1x PBS (50 ml) (see Recipes)
    
29. 1x Trypsin-EDTA (10 ml) (see Recipes)
    
30. 1% Triton X-100 (50 ml) (see Recipes)
    
31. 0.9% saline solution (see Recipes)
    
32. LB medium (100 ml) (see Recipes)
    
33. LB Agar (100 ml) (see Recipes)
    
34. Alkaline phosphatase assay buffer (10 ml) (see Recipes)
    

Equipment

1. 25 cm² tissue-culture treated flasks (BD Biosciences, Falcon^®, catalog number: 353108 )
   
2. Sterile, disposable 15 ml and 50 ml centrifuge tubes
   
3. Sterile, disposable 1.5 ml microcentrifuge tubes
   
4. Sterile microtips
   
5. Sterile cryovials
   
6. Cell scraper (BD Biosciences, Falcon^®)
   
7. 0.22 μm filter units (Millex-GP) (Millipore, catalog number: SLGP033RS )
   
8. CO₂ incubator
   
9. Laminar flow hood
   
10. Liquid nitrogen storage container
    
11. Inverted microscope
    
12. Centrifuge
    
13. Microcentrifuge
    
14. Pipetteman
    
15. UV-Vis Spectrophotometer
    

Procedure