Materials and Reagents

1. Sterile single-use inoculating loop
   
2. 200 μl pipette tips (such as: Corning, Axygen^®, catalog number: # T-200-C )
   
3. Parafilm (Bemis, catalog number: PM996 )
   
4. Syringe-mounted 0.45-µM filter (EMD Millipore, catalog number: SLHV033RS )
   
5. Polyethersulfone (PES) filter, 0.45-µM, Ø75-mM membrane diameter, with an attached 500-ml bottle. Operated by a vacuum pump (e.g., Guangzhou Jet Bio-Filtration, catalog number: FPE404500 )
   
6. 1 ml and 10 ml sterile syringes (without needle)
   1 ml syringes (such as: KDL Medical Product, catalog number [as written on the package]: 7290010993604)
   10 ml syringes (such as: Medisposables, catalog number [as written on the package]: 9304-10/16933506093041 )
   
7. Sterile 15-ml Falcon tube (such as: SPL Life Sciences, catalog number: 50015 )
   
8. 12 ml polypropylene/polyallomer ultracentrifuge tubes (such as: Beckman Coulter, catalog number: 331372 ) – make sure that they are fit for the ultracentrifuge’s speed
   
9. Syringe-mounted 0.22-µM filter (Axiva Sichem Biotech, catalog number: SFPV13 R )
   
10. Sterile 1.5-ml tube (1.7-ml clear tube) (Corning, Axygen^®, catalog number: # MCT-175-C )
    
11. 1 ml pipette tips (such as: Corning, Axygen^®, catalog number: # T-1000-C )
    
12. Polystyrene (PS) aseptic Petri dishes, 90 x 15-mm (Miniplast Ein-Shemer, catalog number: 820-090-01-017 )
    
13. Xanthomonas strain in glycerol stock stored at -80 °C – for the preparation of this protocol we used X. campestris pv. campestris (Xcc) 33913, which was obtained from American Type Culture Collection (ATCC)
    
14. Difco nutrient agar (BD, catalog number: 21300 )
    Note: It contains 3 g/L of beef extract, 5 g/L of peptone, and 15 g/L of agar, with a final pH of 6.8 ± 0.2 (as written on the powder’s container).
    
15. Cephalexin hydrate (Sigma-Aldrich, catalog number: C4895 ) – stock solution at concentration of 10 mg/ml
    
16. Sterile dH₂O
    
17. 10x phosphate buffered saline (PBS) buffer (optional) (such as: Sigma-Aldrich, catalog number: P5493 )
    
18. OptiPrep density gradient medium (Sigma-Aldrich, catalog number: D1556 )
    
19. ‘Coomassie Plus – The Better Bradford Assay Kit’ (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 23236 ) (optional)
    
20. Lipid dye FM4-64FX (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: F34653 ) (optional)
    
21. Materials and reagents required for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as indicated in the following protocol (optional): http://www.bio-protocol.org/e80 (‘[Bio101] Laemmli-SDS-PAGE’)
    
22. Yeast extract powder (EMD Millipore, catalog number: 61931105001730 )
    
23. Bacto tryptone (BD, Bacto, catalog number: 211705 )
    
24. Sodium chloride (NaCl) (EMD Millipore, catalog number: 7647-14-5 )
    
25. Sucrose crystallized (Duchefa Biochemie, catalog number: S0809 )
    
26. Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (EMD Millipore, catalog number: 105886 )
    
27. NaOH or HCl (in order to adjust pH)
    
28. Peptone (EMD Millipore, catalog number: 61930705001730 )
    
29. L(+)-glutamic acid (Acros Organics, catalog number: 6106-04-3 )
    
30. HEPES (Sigma-Aldrich, catalog number: H3375 )
    
31. YEB medium (see Recipes)
    
32. PSB medium (see Recipes)
    
33. OptiPrep diluent buffer (see Recipes)
    

Note: (optional) – not necessary for the vesicles’ isolation process.

Equipment

1. Biological hood
   
2. Temperature controlled incubator shaker (28 °C), with a capability to reach 200 RPM (such as: Thermo Fisher Scientific, Thermo Scientific^TM, model: MaxQ^TM 6000 )
   
3. Two 2-L Erlenmeyer flasks
   
4. Cuvette 
   
5. Polypropylene Copolymer (PPCO) 250-ml centrifuge bottles (such as: Thermo Fisher Scientific, Thermo Scientific^TM, model: Nalgene^TM PPCO Centrifuge Bottle ) – make sure that they are fit for the centrifuge’s speed
   
6. High-speed centrifuge (e.g., Beckman Coulter, model: Avanti J-E , catalog number: 369003; or Thermo Fisher Scientific, model: Sorvall^TM RC-6 , catalog number: 74804^*) and 6 x 250-ml rotor (such as: Beckman Coulter, model: JLA-16.250 , catalog number: 363930, dual-locking lid; or Thermo Fisher Scientific, Sorvall^TM, model: SLA-1500 , catalog number: 46615^* [old model]) at a speed of approx. 32,000 x g
   
7. Ultracentrifuge (e.g., Thermo Fisher Scientific, Sorvall^TM, model: Discovery 90SE , catalog number: 40130867^*, Kendro Laboratory Products) and a 6 x 12-ml swinging bucket ultracentrifuge rotor such as: Thermo Fisher Scientific, Sorvall^TM, model: TH-641 , catalog number: 54295^* (old model, Kendro Laboratory Products), or Thermo Fisher Scientific, Thermo Scientific^TM, model: TH-641 , catalog number: 54295 (new model,) at a speed of approx. 175,000 x g
   
8. Autoclave
   
9. NanoSight device (Malvern) (optional)
   
10. Transmission electron microscopy (optional)
    
11. Equipment required for SDS-PAGE as indicated in the following protocol (optional): http://www.bio-protocol.org/e80 (‘[Bio101] Laemmli-SDS-PAGE’)

Notes

1. *Catalog number of an old model.
   
2. (optional) – not necessary for the vesicles’ isolation process.
   
3. Important note: the centrifuge speeds mentioned above, were calculated with Science Gateway's centrifuge rotor speed calculator (http://www.sciencegateway.org/tools/rotor.htm) – if the centrifuge/ultracentrifuge did not give such option.

Procedure