Sean Lauber:Arginase assay

This procedure is meant to look for arginase activity in macrophages. The higher this activity is, it's believed that this is more of an M2 macrophage. Cells from the BAL can first be adhered to a plate and washed to remove other cell types. This procedure has been modified to work on a 96-well PCR plate (works well).

1) Wash cells with 100 ul PBS

2) Lyse cells in 25 ul of 0.1% Triton X-100 supplemented with protease inhibitors

 For 1 ml inhibitor:		
   5 ul of 200 mM Na3VO4	
   5 ul of 1 mM PMSF	
   1 ul of 0.1 M DTT	
   30 ul aprotonin	

3) Possible to store samples at -80*C at this point

4) Incubate for 15 min on orbital shake to ensure lysis at RT

5) Add 25 ul of 25 mM Tris-HCl (pH 7.5)

 For 50 ml of 25 mM Tris-HCl:		
   50 ml of water	
   0.15 g of Tris	
   pH to 7.5 with HCl	

6) Remove 25 ul and place it into a PCR plate

7) Add 2.5 ul of 10 mM MnCl2

 For 1 ml 10 mM MnCl2:		
   10 ul of 1 M MnCl2	
   990 ul water	

8) Put samples in thermal cycler and put at 56*C for 10 min

9) Add 25 ul of 0.5 M L-arginine

 For 10 ml of 0.5 M L-arg:		
   0.871 g L-arg	
   10 ml water	

10) Incubate at 37*C for 30 min

11) Prepare the urea standard:

 For 200 ml of 10 mM urea:		
   0.12 g urea	
   100 ml water	
   Make a 7-point curve with this (10, 5, 2.5, 1.25, 0.625, 0.3125, 0)	

12) Add 200 ul of acid solution

 For 110 ml of acid solution:		
   70 ml water	
   10 ml H2SO4 (add slowly)	
   30 ml H3PO4 (add slowly)

13) Add 10 ul of 9% alpha-ISPF

 For 1 ml of alpha-ISPF		
   0.09 g alpha-ISPF	
   1 ml 100% EtOH	

14) Incubate at 95*C for 30 min (ensure the caps are securely fastened and will not pop open)

15) Let cool to 20*C and immediately remove the plate

16) Quickly transfer samples to 96-well plate and ensure there is no precipitate in the liquid

 If there is a precipitate the spectrophotometer will not give reliable absorbances. Put the plate back to 95*C to dissolve the precipitate.

17) Read at 550 nm