XTT Assay of Antifungal Activity

Materials and Reagents

1. Aspergillus fumigatus (A. fumigatus) clinical isolate Af293 (Nierman et al., 2005)
   
2. Human pDCs obtained from healthy donors
   
3. RPMI-1640 medium without phenol red (Life Technologies, Gibco^®, catalog number: 11875119 )
   
4. Penicillin G/ streptomycin sulfate (Life Technologies, Gibco^®, catalog number: 15140122 )
   
5. L-glutamine (Life Technologies, Gibco^®, catalog number: 25030081 )
   
6. Sodium pyruvate (Life Technologies, Gibco^®, catalog number: 11360070 )
   
7. Sterile distilled water (Life Technologies, Gibco^®, catalog number: 10977015 )
   
8. Sterile PBS (Corning Incorporated, catalog number: 21040CM )
   
9. 96 well half area plates (Corning, Costar^®, catalog number: 07200309 )
   
10. Tips
    
11. 15 ml Centrifuge tube (Falcon^®，catalog number: 352059 )
    
12. XTT (Sigma-Aldrich, catalog number: X4251 )
    
13. Coenzyme Q (Sigma-Aldrich, catalog number: D9150 )
    
14. Tween 20 (Thermo Fisher Scientific, catalog number: BP337500 )
    
15. Sabouraud dextrose agar (Thermo Fisher Scientific, catalog number: R454472 )
    
16. Supplemented media (see Recipes)
    
17. Slants (see Recipes)
    

Equipment

1. 30 μm Nylon mesh (Genesee Scientific Corporation, catalog number: 57105 )
   
2. 10 ml Syringe (BD Bioscience, catalog number: 305482 )
   
3. Inverted microscope (Nikon)
   
4. Pipettes
   
5. Multichannel pipette
   
6. Neubauer chamber
   
7. Reagents reservoir (Corning, Costar^®, catalog number: 07200127 )
   
8. Microplate reader with capacity to measure OD₄₅₀ and OD₆₅₀ (Versamax)
   
9. Water bath (Thermo Fisher Scientific)
   
10. CO₂ Incubator (37 °C)
    
11. Vortex (Fisher Vortex Genie 2) (Scientisfic Industries, Inc., catalog number: 12812 )
    
12. Autoclavec
    

Procedure