1. Add 200 ul of PBS to tubes with 3x flies and homogenize.

2. Serial Dilute 50 ul of standard.

• 50 ul = 2 ug/ul = Std07

• 25 ul of Std07 + 25 ul PBS = 1 ug/ul = Std06

• 25 ul of Std06 + 25 ul PBS = 0.5 ug/ul = Std05

• 25 ul of Std05 + 25 ul PBS = 0.25 ug/ul = Std04

• 25 ul of Std04 + 25 ul PBS = 0.125 ug/ul = Std03

• 25 ul of Std03 + 25 ul PBS = 0.0625 ug/ul = Std02

• 25 ul of Std02 + 25 ul PBS = 0.03125 ug/ul = Std01

3. Put 10 ul of Blanks (BL = PBS); Standards (Std01-07); and Sample (Sa01-40) into 96-well plate as illustrated below. Each blank/standard/sample has a technical replicate:

4. Add 50 ul of Activator to every 5 ml of Reagent. Invert gently 3-4 times.

• Let sit for 15 min at room temperature before use.

• After 15 min, add 190 ul Activator/Reagent solution to each well of plate.

• 190 ul A/R solution + 10 ul sample = 200 ul total

• Let plate sit at least 10 min (30 min okay) at room temperature.

5. Turn on SpectraMax M2 machine and computer.

6. Press "drawer" button to open/close plate drawer and load your plate.

7. Open SoftMax Pro software. If "no port selected" choose Com1.

• Click 'plate' to read plate, 'cuvette' to read cuvette

• Save as

• Settings

• aborbance

• wavelength: 500 nm

• endpoint

• automix

• OK

• Template

• unknown

• concentration ug/ml

• standards

• 1st blank

• group

• new group setting

• name = sample

• Results

• Result Interpx(Plot#1@Graph#1,Values)

• R² should be ~1

• y = absorbance, x = concentration, B = slope

• y = A+Bx => x = (y-A)/B

• TG_total/corrected fly weight = ug/mg = plot this value