Chitinase Assay from Cultured Bone Marrow Derived Macrophages

Materials and Reagents

1. Cells to be analyzed. e.g. Bone Marrow-derived macrophages from C57BL/6 mice
   
2. L-cell
   
3. cDMEM/F12
   
4. 4-Mehtylumbelliferyl labeled substrates:
   1. 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide (exochitinase activity; β–N-acetylglucosaminidase activity) (Sigma-Aldrich, catalog number: M2133 )
      
   2. 4-Methylumbelliferyl N,N′-diacetyl-β-D-chitobioside (exochitinase activity; chitobiosidase activity) (Sigma-Aldrich, catalog number: M9763 )
      
   3. 4-Methylumbelliferyl β-D-N,N′,N′′-triacetylchitotriose (endochitinase activity) (Sigma-Aldrich, catalog number: M5639 )
      
5. Chitinase from Trichoderma viride (Sigma-Aldrich, catalog number: C6242 )
   
6. Methylumbelliferone Standard (50 mg/ml) (Sigma-Aldrich, catalog number: M1381 )
   
7. PBS
   
8. DMSO
   
9. Plate reader
   
10. Diabasic Sodium Phosphate
    
11. Citric Acid
    
12. Glycine
    
13. DTT
    
14. Assay buffer (see Recipes)
    
15. Stop buffer (see Recipes)
    
16. Protein lysis buffer (see Recipes)
    
17. Substrate Stock (see Recipes)
    
18. Positive control (see Recipes)
    

Equipment

1. Flat bottom black 96-well plates
   
2. Fluorescent plate reader (Molecular Devices)
   

Procedure