Amplite® Fluorimetric Endotoxin Detection Kit

1. Prepare Endotoxin Green™ working solution
2. Add E.coli Endotoxin Standards and test samples (25 µL)
3. Add Limulus Amebocyte Lystate solution (25 µL)
4. Incubate at 37 °C for 30 minutes
5. Add Endotoxin Green™ working solution (50 µL)
6. Read fluorescence intensity at Ex/Em=490/525 nm within 10 minutes

Thaw all the kit components at room temperature before starting the experiment.

All Materials used in the experiment should be endotoxin-free, such as: disposable tubes or 1.5 mL microcentrifuge tubes, disposable pipette tips, and disposable 96-well microplates or plate strips. The cleanliness of all labware is required to accurately detect levels of endotoxin in a given sample.

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 50 µL of DMSO into the vial of Endotoxin Green™ (Component A) to make 100X Endotoxin Green™ stock solution.
Note        Keep from light.

Add 500 µL of Endotoxin-Free Water (Component B) into the vial of Limulus Amebocyte Lysate (Component C) to make 5X Limulus Amebocyte Lysate solution.

Add 50 µL of Endotoxin Green™ stock solution into 5 mL of Endotoxin-Free Water (Component B) to make a total volume of 5.05 mL Endotoxin Green™ working solution.
Note        Prepare the amount of Endotoxin Green™ working solution as needed. Keep the working solution from light.

Add 500 µL of  Limulus Amebocyte Lysate (LAL) Stock Solution into 2 mL of Endotoxin-Free Water (Component B) to make a total volume of 2.5 mL Limulus Amebocyte Lysate (LAL) working solution.
Note        Prepare the amount of LAL working solution as needed and before use. Using the Endotoxin-Free bottle or tube.

Table 1. Layout of E.coli Endotoxin Standards and test samples in a solid black 96-well microplate. ES=E.coli Endotoxin standards (ES1-ES7, 1.00 to 0.001 EU/mL); BL=Blank Control; TS=Test Samples

Table 2. Reagent composition for each well.

1. Prepare E.coli Endotoxin Standards (ES), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 12.5 µL of reagent per well instead of 25 µL.

2. Add 25 µL of 1X Limulus Amebocyte Lysate solution to each well of E.coli Endotoxin Standard, blank control and test samples.

3. Mix well and incubate for 30 minutes at 37 °C.
4. Add 50 µL of Endotoxin Green™ working solution to each well of E.coli Endotoxin Standard, blank control, and test samples to make the total assay volume 100 µL/well. For a 384-well plate, add 25 µL of Amplite™ Endotoxin Green working solution into each well instead, for a total volume of 50 µL/well.
5. Monitor the fluorescence intensity at Ex/Em=490/525 nm, cutoff 515 nm.
   
   Note        For best results, read between 2 to 10 minutes after adding the working solution.
   
   Note        25 µL of 25% acetic acid can be added to stop the reaction.