LUMIstar Omega

GPCRs are important drug targets requiring receptor-protein interaction and trafficking studies to reveal how they function. Bioluminescence resonance energy transfer (BRET) is a versatile tool to study such interactions and trafficking. Hitherto, it is limited by the ectopic expression of labelled interaction partners. CRISPR/Cas9 genome editing overcomes the limitation by enabling endogenous expression of luciferase-labelled proteins.

CRISPR/Cas9-edited cells endogenously expressing a CXCR4/NanoLuciferase fusion protein were used in conjunction with β-Arrestin/Venus to monitor receptor activation. Employing two fluorophores fused to a membrane and endosome standing CXCR4-interacting protein, respectively, allowed for monitoring of receptor trafficking.

The novel CRISPR/Cas9 technique successfully fused the Nluc BRET donor to endogenously-expressed CXCR4. The resulting protein levels were sufficient to monitor receptor interactions as well as internalization. The internalization assay depends on two acceptor fluorophores whose selective detection was rendered possible by the CLARIOstar’s monochromator.

Schistosomiasis is a parasitic disease that affects over 200 million people in tropical, developing nations, causing severe morbidity and over 300,000 deaths annually. Schistosomiasis is treated with a single drug and no vaccine is available.

We selected three Schistosoma surface-associated enzymes that are indispensable to parasitic survival: alkaline phosphatase; phosphodiesterase SmNPP-5 and an acetylcholinesterase. The activity of these molecules on the surface of live and intact larval and adult Schistosoma can be assayed in real-time of cultured parasites, providing a tool to assess the efficacy of drugs or vaccines targeting these enzymes. The colorimetric assay was read on a FLUOstar^® Omega microplate reader.

The versatile instrument supports development of assays and drugs. Apart from its absorbance spectrometer, the FLUOstar Omega is equipped with fluorescence and luminescence detection capabilities allowing fast and reliable endpoint and kinetic measurements in all detection modes.

SPARCL refers to an immunoassay that does not require washing steps. An analyte in a solution is detected by binding two antibodies: one is coupled to horse radish peroxidase (HRP), the other is coupled to the HRP substrate Acridan. In the presence of the analyte, binding of both antibodies brings the enzyme and substrate into proximity. The enzymatic reaction is started by the addition of H₂O₂, which immediately produces a light proportional to the amount of analyte. Thus, SPARCL assays require to be read at the same time as the injection of the trigger solution.

The CLARIOstar^® microplate reader, which is equipped with the dedicated H3 injection needle for simultaneous injection and reading, fulfils this task. The analyte IgG was detected down to a concentration of 45 ng/ml in 96 wells and down to 15.6 ng/ml in 384 well format. BMG LABTECH's MARS Data Analysis Software facilitates subsequent calculation of analyte concentration.

Detection of biomarker interleukin 8 (IL-8) is a good measure to evaluate the differences of normal versus pathogenic processes in the human body. Here we will show how the SPARCL technology can be used in combination with the BMG LABTECH microplate reader instrumentation to detect IL-8 as well as IgG.
 

The SPARCL technology is based on a specific antibody/antigen interaction that brings acridan and HRP into close proximity. After addition of a hydrogen peroxide trigger solution a flash of light follows. The luminescent flash exists only for a very limited time. A typical SPARCL pattern of flash luminescence shows that most of the signal is already gone after 1 second. Because of this situation it is necessary to use an instrument that is able to inject trigger solution and measure flash luminescence at the same time.

© 2015 Lumigen, Inc. All rights reserved. Used with permission. Lumigen and SPARCL are trademarks of Lumigen, Inc. and are registered in the USPTO. Lumigen is a Beckman Coulter company.

Mycoplasma are small-sized bacteria that are well-known as contaminant of laboratory cell cultures. Without having a cell wall around their membrane they cannot be targeted with antibiotics. That makes them a big problem and often the whole culture is lost.
 

As mycoplasma can influence experimental results obtained for a certain cell line, cell culture labs need to check regularly if their cultures are contaminated by mycoplasma species. Here we show the application of the MycoAlert™ assay from Lonza to perform this test. The assay is specific for mycoplasmal enzymes that are not present in eukaryotic cells. After addition of substrate a high luminescent signal is created only in viable mycoplasma.
 

The luminescence detection was done in a CLARIOstar^® microplate reader. The instrument comes with the MARS Data Analysis software that allows the user to do calculations and validations to quickly identify samples which may be contaminated.