Amplite® Colorimetric L-Lactate Dehydrogenase (LDH) Assay Kit

1. Prepare L-Lactate Dehydrogenase standards or test samples (50 µL)
2. Add L-Lactate Dehydrogenase working solution (50 µL)
3. Incubate at room temperature for 30 min - 2 hours
4. Monitor absorbance ratio increase at A_575nm/A_605nm

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

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

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

Add 100 µL of H₂O or 1x PBS buffer into the vial of L-Lactate Dehydrogenase (Component D) to make 100 U/mL L-LDH standard solution.

1. Add 10 mL of Assay Buffer (Component B) into the bottle of Enzyme Mix (Component A), and mix well.

2. Add 100 µL of 100X NAD stock solution into the bottle of Component A+B and mix well to make L-LDH working solution.
   
   Note        This L-LDH working solution is enough for two 96-well plates. It is unstable and should be used promptly within 2 hours. Avoid exposure to light. 
   
   Note        Alternatively, one can make a 50X of L-LDH Enzyme Mix stock solution by adding 200 μL of H2O into the bottle of Enzyme Mix (Component A), and then prepare the L-LDH working solution by mix the stock solution with Assay Buffer (Component B) and 100X NAD stock solution proportionally.

Table 1. Layout of L-LDH standards and test samples in a white clear bottom 96-well microplate. SD= L-LDH Standards (SD1 - SD7, 0.4 to 300 mU/mL), BL=Blank Control, TS=Test Samples.

Table 2. Reagent composition for each well.

1. Prepare L-LDH 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 L-LDH working solution to each well of L-LDH standard, blank control, and test samples to make the total L-LDH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of L-LDH 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 absorbance ratio increase with an absorbance plate reader at A_575nm/A_605nm.