Flow Cytometry of Lung and Bronchoalveolar Lavage Fluid Cells from Mice Challenged with Fluorescent Aspergillus Reporter (FLARE) Conidia

Materials and Reagents

1. Cell strainers - 40 μm and 100 μm
   
2. Syringes
   
3. Injector (Modified precision dispensing tips) (Nordson, catalog number: 7018166 )
   
4. 15 ml tubes
   
5. 96-well plate, U-bottom
   
6. Mice: C57BL/6 (Jackson Laboratories, catalog number: 000664 )
   
7. Fungal strains (Af293 and Af293-DsRed) (Jhingran et al., 2012)
   
8. FATAL-plus solution (Vortech Pharmaceuticals Ltd, National Drug Code: 0298-9373-68 )
   
9. BD BBL^TM Sabouraud dextrose agar (Emmons), slants (pH 6.9) (100/sp) (BD, catalog number: 221827 )
   
10. PBS (Thermo Fisher Scientific, Gibco^TM, catalog number: 14190-144 )
    
11. Tween 20
    
12. Tris base
    
13. NaHCO₃
    
14. Na₂CO₃
    
15. Biotin-XX, SSE (6-((6-((biotinoyl)amino)hexanoyl)amino) hexanoic acid, sulfosuccinimidyl ester, sodium salt) (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: B6352 )
    
16. DMSO
    
17. Streptavidin, Alexa Fluor^® 633 conjugate (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: S-21375 )
    
18. Isothesia^TM, Isoflurane (Henry Schein Animal Health, SKU number: 029405 )
    
19. RBC lysis buffer (Biolegend, catalog number: 420301 )
    
20. DNase I (Roche Diagnostics, catalog number: 10104159001 )
    
21. Collagenase type IV (Worthington Biochemical, catalog number: LS004189 )
    
22. EDTA
    
23. Mouse BD Fc Block^TM (BD, BD Pharmingen^TM, catalog number: 553142 )
    
24. Antibodies (listed in Procedure section)
    
25. PBS Tween (0.025%) (see Recipes)
    
26. Tris-chloride (1 M, pH 8) (see Recipes)
    
27. Sodium bicarbonate buffer (0.1 M, pH 9.5) (see Recipes)
    
28. Lung digestion buffer (for each lung) (see Recipes)
    
29. FACS buffer (see Recipes)

Equipment

1. Hemocytometer
   
2. Microscope
   
3. Centrifuge
   
4. Plexiglass stand
   
5. Dissection scissors and forceps
   
6. Bronchoalveolar lavage fluid (BALF) catheters (Exel International, catalog number: 26738 )
   
7. 4-way large bore (lipid resistant) stopcock with rotating male luer lock adapter (Baxter, catalog number: 2C6204 )
   
8. gentleMACS tissue dissociator, MACSMix tube rotator and C tubes (Miltenyi Biotech)
   
9. Automated cell counter or hemocytometer
   
10. Flow cytometer capable of analyzing at least 7 fluorescent parameters (such as BD LSRII). The 532 or 561 nm (but not 488 nm) laser excites DsRed fluorescence and the 633 nm laser excites Alexa Fluor 633 fluorescence.
    

Procedure