You will assess macrophage activation by assaying for nitrit

You will assess macrophage activation by assaying for nitrites, which is indicative of total nitric oxide production. Griess reagent will be added to your samples to obtain an absorbance value that correlates to the number of nitrites present. Griess reagent is made up of reagent A(0.1% naphthalene demine, powder - In dH2O) and reagent B (1% sulfanilamide; powder - in 5% H3PO4). Show how you would make up a 200 mL solution of reagent A and a 200 ml solution of reagent 8. The stock solution is only 85% H3P04 as a stock solution. You can use dH2O as a diluents. You need to make 5O mL of sodium nitrate (FW:84.99) and SO mL of sodium nitrite (FW: 69.0) at a 200 mu M Concentration In dH2O as stock solutions for the standard curve. Describe how you would do this? Please show calculations We need (8 serial) 1/2 dilutions for the standards. Diagram the dilution procedure and the exact concentration of the standard for each tube. The concentration for your stock tube Is 200 uM. We need at least 0.6 mL of each standard. The tubes hold a maximum of 1.5 mL.

Solution

(1)

Preparation of 200 mL of 0.1% naphthylene diamine = 0.2 g of naphthylene diamine dissolved in 100 mL of H2O

Preparation of 200 mL solution 1% sulfanilamide :

We have 85% stock solution of H3PO4

We need 200 mL of 5% H3PO4,

Stock = 85%

Required stock = 5%

Volume = 200 mL

C1V1= C2V2

(stock)   (required)

V1=C2V2/C1

V1 = (5% X 200 mL )/ 85%

=11.7 mL

11.7 mL of stock H3PO4 + 188.3 mL of H2O gives 200 mL of 5% H3PO4

Now dissolve 2g sulfanilamide in 5% H3PO4.