Category: Assay Development and Screening
Inflammation is a complex event in which cells respond to various endogenous and exogenous stimuli. Factors such as tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interferon gamma (IFN-γ) activate signaling pathways leading to the expression of cell-surface antigens that facilitate binding of immune cells to blood vessels. The ability to monitor up-regulation of molecules, such as MCP-1, IL-8, IL-6, VCAM, and HLADR with endothelial cells provides an important physiological read-out for cell-based models of inflammation. We present results from a multiparametric primary human cell-based assay that uses immunoassays for secreted cytokines and fluorescence read-outs of cell surface markers to evaluate the effect of different mediators on inflammatory response. Expression of the inflammation markers on primary human umbilical vein endothelial cells (HUVEC) stimulated with inflammation cytokines (TNF-α, IFN-γ, and IL-1β) was quantified by measurement of total fluorescence intensity after staining with directly conjugated antibodies and microfluidic-based ELISAs. A newly developed microfluidic-based assay system, PuMA Flowchip System, was used to quantify MCP-1, IL-8, and IL-6 in cell supernatants. Complete ELISA protocols (capture Ab to substrate) were run in <3 hrs using 20 µl of each reagent. Absorbance at 450 nm was read using a plate reader (SpectraMax i3). This format provides significant advantages over a multi-well plates by reducing assay times and reagent usage without requiring any antibody conjugation. This enhances to ability to measure multiple cytokines from a single well for inflammation assays where supernatant volume is limited. The system allows running of most antibody pairs providing flexibility from analysis of standard cytokines to novel or proprietary markers. Expression of VCAM and HLADR was quantified by measurement of total fluorescence intensity after staining with directly conjugated antibodies. Images were acquired on an ImageXpress Nano automated imaging system and analyzed with Cell Scoring module in CellReporterXpress automated imaging acquisition and analysis software. Multiparametric inflammation response was characterized as a function of cytokine concentration after 20 hours of stimulation. Concentration dependent effects on inflammation response by the anti-inflammatory compounds AG126, SB202190, MG132, and PDTC was measured. Clear differences in cytokine expression were seen between the compounds consistent with reported mechanisms of actions of the compounds. This novel combination of imaging and microfluidic-based assays provides an efficient multiparametric assay system that can be used to test the efficacy of anti-inflammatory compounds versus toxicity and also provide significant insight into the mechanism of action by selective inhibition of markers triggered by different signaling pathways.
Evan Cromwell– President, Protein Fluidics, Inc., Burlingame, CA
Protein Fluidics, Inc.
Evan F Cromwell, PhD is President & CEO of Protein Fluidics, Inc. a bio-analytical system company located in Burlingame CA in the heart of the San Francisco Bay Area. He has over 30 years of senior executive and technical expertise in numerous hi-tech industries. He has piloted the design, development, and manufacture of systems for biological assays, materials analysis, optical inspection, and process control. Prior to founding Protein Fluidics, he founded and served as the President of Blueshift Biotechnologies. This high-content screening and analysis venture was acquired by Molecular Devices in 2008. After the acquisition, Evan remained onboard at Molecular Devices, which became part of Danaher Life Sciences, where he further honed his business and entrepreneurial acumen. In addition to co-founding successful start-ups, Dr. Cromwell has chaired several scientific advisory boards and led global technology development initiatives. Evan holds a B.S. in Chemistry from Caltech, and earned his Ph.D. in Physical Chemistry from the University of California, Berkeley. He has authored 40+ publications in the fields of biology, physical chemistry, and optical instrumentation and holds 15 patents.