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189 DEVELOPMENT OF A HIGH SENSITIVITY, HIGH THROUGHPUT SCREEN FOR NEUROINFLAMMATORY RESPONSE USING A MOLECULAR MARKER AND MICROGLIAL ACTIVATION
  1. M. Sexton1,
  2. D. J. Bornhop2,
  3. N. Stella1,
  4. M. Bai2
  1. 1Bastyr University, Seattle, WA
  2. 2Vanderbilt University

Abstract

Introduction Microglial cells are mononuclear macrophage cells unique to the Central Nervous System (CNS). The peripheral benzodiazepine receptor (PBR) is a mitochondrial membrane protein known to be involved in a variety of cellular functions. There is a correlation between upregulated PBR expression and aberrant cell growth, proliferating cancer and neuroinflammation.

Purpose Here we demonstrate the potential for employing a high throughput plate based assay as a screening tool. By targeting the PBR using a fluorescently labeled PBR ligand (RED-PK11195), a plate-based assay was developed using microglial cells labeled with RED-PK and fluorescence was measured in response to various stimuli.

Methods The murine microglial cell line BV2 was cultured and plated in 24 well plates in MEM/Cellgro, incubated over night with various immunomodulatory factors, subsequently rinsed and labeled with the RED-PK dye in PBS. Fluorescence values were measured on a plate reader and PBR expression quantified. As in the past, microglial activity appears to be correlated with PBR expression as quantified using the fluorescent dye.

Summary BV2 cells were capable of being labeled with the RED-PK dye as confirmed with fluorescence microscopy. BV2 cells were capable of producing a change in PBR expression under varying conditions and fluorescence values could be quantified on a plate reader.

Conclusions PBR is a viable target for molecular labeling in microglial cells, providing an indicator of activation status. Potential applications for this high throughput screen using a plate based assay are numerous. Ongoing studies will evaluate the correlation of PBR expression with cytokine, nitric oxide and reactive oxygen species (ROS) production.

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