Portelite™ Rapid Fluorimetric Endotoxin Detection Kit

1. Prepare Limulus Amebocyte Lysate (LAL) working solution
2. Add E.coli Endotoxin Standards and test samples (50 µL)
3. Add Limulus Amebocyte Lysate working solution (50 µL)
4. Incubate at 37 °C for 30 minutes
5. Prepare and add Endotoxin Green™ working solution (100 µL)
6. Monitor fluorescence with CytoCite™ Fluorometer 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 tubes. 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

For kit# 60008, add 50 µL of DMSO into one vial of Endotoxin Green™ (Component A) to make Endotoxin Substrate stock solution. For kit# 60009, add 500 µL of DMSO into one vial of Endotoxin Green™ (Component A) to make Endotoxin Substrate stock solution.
Note        Keep from light.

For kit# 60008, add 500 µL Endotoxin-Free Water (Component B) to the vial of Limulus Amebocyte Lysate (Component C) to make 5X  Limulus Amebocyte Lysate (LAL) stock solution. For kit# 60009, add 2.5 mL Endotoxin-Free Water (Component B) to the vial of Limulus Amebocyte Lysate (Component C) to make 5X Limulus Amebocyte Lysate (LAL) stock solution.

For Kit# 60008, 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. For Kit# 60009, add 500 µL of Endotoxin Green™ stock solution into 50 mL of Endotoxin-Free Water (Component B) to make a total volume of 50.5 mL Endotoxin Green™ working solution.
Note        Each test needs 100 µL, please prepare the amount of Endotoxin Green™ working solution as needed and before use. Please also keep the working solution from light after preparation, and use Endotoxin-Free bottle or tube and store unused stock solutions at -20 °C.

For kit# 60008, 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. For kit# 60009, add 2.5 mL of Limulus Amebocyte Lysate (LAL) stock Solution into 10 mL of Endotoxin-Free Water (Component B) to make a total volume of 12.5 mL Limulus Amebocyte Lysate (LAL) working solution.
Note        Each test needs 50 uL, please prepare the amount of LAL working solution as needed and before use. Please also keep the working solution from light after preparation, use Endotoxin-Free bottle or tube, and store unused stock solutions at -20 °C.

1. Prepare E.coli Endotoxin Standards, blank controls or test samples (50 µL) in a 0.2 mL PCR tube (Cat# CCT100).
2. Add 50 µL of Limulus Amebocyte Lysate working solution to each tube of E.coli Endotoxin Standard, blank control and test samples.
3. Mix well and incubate for 30 minutes at 37 °C.
4. Add 100 µL of Endotoxin Green™ working solution to each tube of Endotoxin Standard, blank control, and test samples to make the total assay volume 200 µL/well.
5. Insert the samples into CytoCite™ and monitor the fluorescence with green fluorescent channel. Follow the procedure appropriate for CytoCite™ Fluorometer. See the link below for detailed instructions: https://devices.aatbio.com/documentation/user-manual-for-cytocite-fluorometer.
   
   Note        For best results, read between 2 to 10 minutes after adding the working solution.
   
   Note        50 µL of 25% acetic acid can be added to stop the reaction.