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High Throughput, Multiplexed Detection of Inflammatory Cytokines in an Astrocyte and Monocyte Co-culture Model

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February 04, 2016

Authors: Brad Larson, BioTek Instruments, Inc. Winooski, VT USA; Roger Bosse, PerkinElmer, Waltham, MA USA, and Tracee Crossett, Lonza Group, Ltd., Basel Switzerland

PerkinElmer     Lonza

 

Abstract

Inflammatory cytokine levels have been reported to undergo significant increase in multiple neurodegenerative disorders, including Alzheimer’s disease (AD) and multiple sclerosis (Mrak, et al., 1995). It has been repeatedly documented that astrocytes are a critical source of IL-6, while glial cells are an important producer of IL-1 and TNFα (Chao, et al., 1995). Research has also revealed that under certain conditions, astrocytes and neurons can express multiple different cytokines which are normally undetectable. These three cell types commonly interact to produce the inflammatory response. For example, in AD activated astrocytes and microglia are characteristically found in abundance near neurons and plaques (Griffin, et al., 1989). Therefore it is imperative that assessment of pertinent cytokine levels be performed with relevant cell types in isolation as well as in a co-culture model.

In the past, cytokine levels were assessed using assays that measured protein concentrations on an individual basis. As cytokines are secreted into the surrounding media, multiple analyses are still able to be performed from a single well. However, the procedure is labor intensive and performing individual assessments in separate wells can increase variability within the collective data set. By incorporating an assay technology capable of providing assessment of multiple cytokines in the same well, variability can be eliminated, creating a robust detection method.

Here we describe a homogeneous, bead-based immunoassay technology which offers multiplexed detection of several cytokines in a single well. Donor beads are bound to antibodies specific for one of the target analytes being assessed. Acceptor beads, providing disparate emission profiles upon excitation, are also directly conjugated to a specific anti-analyte antibody. In the presence of the individual analyte, the beads come into close proximity to each other. When excited using the 680 nm laser on a novel high throughput multi-mode microplate reader, donor beads convert dissolved oxygen to singlet oxygen molecules, causing a cascade of energy transfer in the acceptor beads and an increase of light emission at the appropriate wavelength. Therefore high signal values are indicative of greater cytokine emission within the well. Cryopreserved primary astrocytes and peripheral blood CD14+ monocytes were incorporated independently and in co-culture to increase the relevance of generated results. The combination of multiplexed assay technology, cell model and laser-based detection provide a rapid, yet highly sensitive method to assess levels of important inflammatory cytokines.

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