HEK-Blue™ LPS Detection Kit

Lipopolysaccharide (LPS), also known as endotoxin, the major cell wall component of Gram-negative bacteria, induces the activation of NF-κB and the production of proinflammatory cytokines.
In vivo, this response can cause fever, septic shock and eventually death of the animal. In vitro, it can introduce a bias in experiments involving cells sensitive to low levels of LPS such as monocytes.
In addition, repeated passages of cell lines in a medium containing LPS might render these cells unresponsive to further stimulation by LPS. This desensitization, termed LPS tolerance, does not only affect inflammatory responses but also other essential functions including antigen presentation by monocytes. Thus, monitoring the presence of LPS in biological reagents is crucial.
InvivoGen provides the HEK-Blue™ LPS Detection Kit, a simple, rapid and reliable system to detect the presence of endotoxins in your samples.

HEK-Blue LPS Detection Kit Procedure

The HEK-Blue™ LPS Detection Kit is based on the ability of TLR4 to recognize structurally different LPS from gram-negative bacteria and in par ticular lipid A, their toxic moiety.
Proprietary cells engineered to become extremely sensitive to LPS, called HEK-Blue™-4 cells, are the main feature of this endotoxin detection kit. The presence of very low concentrations of LPS, starting as low as 0.03 ng/ml, are detected by the HEK-Blue™-4 cells leading to the activation of NF-κB.
Using HEK-Blue™ Detection, a specific detection medium, NF-κB activation can be observed with the naked eye or quantified by reading the OD at 650 nm.

LPS Detection using the HEK-Blue™ LPS Detection Kit
The presence of LPS in various preparations of TLR ligands was determined using the HEK-Blue™ LPS Detection Kits: 1) Monophosphoryl lipid A, 2) LPS-EB (E. coli 0111:B4), 3) LPS-EK (E. coli K12), 4) LPS-SM (S. minnesota), 5) LPSPG (P. gingivalis), 6) ODN 2006, 7) E. coli DNA ef (endotoxin-free), 8) E. coli DNA we (with endotoxin).

HEK-Blue™-4 cells, the endotoxin sensor cells, are engineered HEK293 cells. These cells stably express TLR4, MD2, CD14 and multiple genes from the TLR4 pathway. They coexpress an optimized SEAP repor ter gene, placed under the control of a promoter inducible by the transcription factors NF-κB and AP-1.

HEK-Blue™ Selection is a solution that combines several selective antibiotics. These antibiotics guarantee the persistent expression of the various transgenes introduced in HEK-Blue™-4 cells. Fur thermore, Normocin™ is included in the kit to protect HEK-Blue™-4 cells from any potential microbial contamination, whether caused by mycoplasmas, bacteria or fungi.

HEK-Blue™ Detection is a medium specifically designed for the detection of SEAP. It contains a color substrate that produces a purple/blue color following its hydrolysis by SEAP.

The HEK-Blue™ LPS Detection Kit is composed of the following components:
- 1 vial of HEK-Blue™-4 cells (3-5x 106 cells)
- 4 tubes of 250X HEK-Blue™ Selection (2 ml)
- 4 tubes of 500X Normocin™ (1 ml)
- 2 pouches of HEK-Blue™ Detection (50 ml each)
- 1 tube of E. coli K12 LPS, LPS-EK, (100 μg) as a positive control
- 1 tube of endotoxin-free water (1.5 ml) as a negative control
Buy the HEK-Blue™ LPS Detection Kit once then reorder only the reagents to perform further assays.