Amplite® Fluorimetric D-Lactate Assay Kit

Protocol summary

1. Prepare D-Lactate working solution (50 µL)
2. Add D-Lactate standards or test samples (50 µL)
3. Incubate at room temperature for 30 min - 2 hours
4. Monitor fluorescence increase at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Important notes
Thaw one vial of each kit component at room temperature before starting the experiment.

1. NAD stock solution (100X):
Add 100 µL of H₂O into the vial of NAD (Component C) to make 100X NAD stock solution.

2. D-Lactate standard solution (100 mM):
Add 200 µL of H₂O or 1x PBS buffer into the vial of D-Lactate Standard (Component D) to make 100 mM D-Lacate standard solution.

1. Add 5 mL of Assay Buffer (Component B) into one bottle of Enzyme Mix (Component A), and mix well.

2. Add 50 µL of 100X NAD stock solution into the bottle of Component A+B, and mix well to make D-Lactate working solution. Note: This D-Lactate working solution is enough for one 96-well plate. Protect from light.

Table 1. Layout of D-Lactate standards and test samples in a solid black 96-well microplate. SD= D-Lactate Standards (SD1 - SD7, 1 to 1000 µM), BL=Blank Control, TS=Test Samples. 

Table 2. Reagent composition for each well.

1. Prepare D-Lactate standards (SD), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
   
   
2. Add 50 µL of D-Lactate working solution to each well of D-Lactate standard, blank control, and test samples to make the total D-Lactate assay volume of 100 µL/well. For a 384-well plate, add 25 µL of D-Lactate working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction at room temperature for 30 minutes to 2 hours, protected from light.
   
   
4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm, Cutoff = 570 nm).