In vitro Analysis for Macrophage Binding and Pro-inflammatory Responses to Candida albicans

Materials and Reagents

1. Candida albicans strains, SC5314 and ATCC18804
   
2. Mice
   Note: 2x per WT or Dectin-1 KO mice on C57BL/6 background should give sufficient macrophages for a full 24-well plate experiment. Mice were age and sex matched.
   
3. Thioglycollate-elicited macrophages
   Note: This method has been described in detail in Kerrigan et al. (2012).
   
4. RPMI 1640 medium (Life Technologies, catalog number: 11875-093 ) 
5. 70% ethanol
6. Sabouraud Dextrose broth (Oxoid Limited, catalog number: CM0147 )
   
7. Rhodamine Green-X (Life Technologies, Invitrogen^TM, catalog number: R-6113 )
   
8. Difco Thioglycollate Broth (BD Biosciences, catalog number: 225710 )
   
9. Triton-X 100 (Sigma-Aldrich, catalog number: T8787 )
   
10. OptEIA TNF kit (BD Biosciences, catalog number: 555268 )
    
11. Zymosan (Life Technologies, Invitrogen^TM, catalog number: Z2849 )
    
12. Dulbecco’s Phosphate Buffered Saline (PBS) (Sigma-Aldrich, catalog number: D8662 )
    
13. Fetal Bovine/Calf Serum (FCS) (Sigma-Aldrich, catalog number: F6178 )
    
14. EDTA (Sigma-Aldrich, catalog number: E9884 )
    

Equipment

1. Titer-Tek Fluoroskan II (Labsystems)
   
2. Shaker
   Note: The shaker temperature was controlled at 30 °C and the speed was between 150 to 200 rpm.
   
3. Centrifuge (Eppendorf, model: 5810R with swing bucket rotor S-4-72 )
   
4. Light microscope
   
5. Haemocytometer
   
6. 24-well plates (BD Biosciences)
   
7. 2 ml Eppendorf tube
   
8. 96-well plates (black in color for fluorescence measurement) (BD Biosciences)
   
9. Rubber back of syringe plunger
   Note: The make or brands for above equipment are not important for reproducibility.
   

Procedure