Investigating the effects of membrane-bound CX3CL1 on microglial migration using alternative transgenic models
Poster #: 137
Session/Time: B
Author:
Austin David Grove, BS
Mentor:
Jennifer Ness-Myers, PhD
Research Type: Basic Science
Abstract
INTRODUCTION:
Fractalkine (CX3CL1, FKN) is a transmembrane chemokine produced by neurons in the central nervous system (CNS). It binds to its respective receptor (CX3CR1), which is expressed on the surface of microglia, the innate immune cells of the CNS. Fractalkine plays an important role in activating pro-inflammatory pathways in microglia in response to tissue injury. This signaling is elicited by both the soluble (cleaved) form and the membrane-bound form of fractalkine. The soluble form of fractalkine is released from the neuronal surface following neuronal injury, which elicits a pro-migratory response in microglia. The neuron-microglial communication through the membrane-bound form is less well understood. The purpose of this study is to develop a cell culture model to compare microglial migration and signaling responses to membrane-bound and soluble fractalkine.
METHODS:
We transfected 293FT cells (a human kidney cell line) and B6/scl-7 cells with a rat CX3CL1-OFPSpark (C-terminal tag) expression plasmid (Sinobiologicals) to express membrane-bound fractalkine. Primary rat microglia were co-cultured with transfected 293FT cells, B6/scl-7 cells, or treated with soluble rat fractalkine (Peprotech).
RESULTS:
293FT cells were successfully transfected using Fugene 6®, but B6/scl-7 cells were not. Alternatively, B6/scl-7 cells were electroporated using Neon® Transfection System, but resulted in cell death. Microglia plated with FKN-expressing 293FT cells interacted favorably with the transfected cells, while those plated on untransfected 293FT cells exhibited unfavorable interactions and were rounded.
CONCLUSION:
These findings suggest transfecting B6/scl-7 cells with another method such as Lipofectamine 3000®. These models interact favorably with microglia compared to untransfected controls, showing these are viable models. Once stable transfections are achieved for both cell lines, these models will be used to: visualize cell-cell interactions of microglia in response to fractalkine signaling, quantify these interactions with Boyden chamber migration assays and CyQuant GF Fluorescent dye, and observe the regulation of NFκB in the signaling pathway of fractalkine.
Fractalkine (CX3CL1, FKN) is a transmembrane chemokine produced by neurons in the central nervous system (CNS). It binds to its respective receptor (CX3CR1), which is expressed on the surface of microglia, the innate immune cells of the CNS. Fractalkine plays an important role in activating pro-inflammatory pathways in microglia in response to tissue injury. This signaling is elicited by both the soluble (cleaved) form and the membrane-bound form of fractalkine. The soluble form of fractalkine is released from the neuronal surface following neuronal injury, which elicits a pro-migratory response in microglia. The neuron-microglial communication through the membrane-bound form is less well understood. The purpose of this study is to develop a cell culture model to compare microglial migration and signaling responses to membrane-bound and soluble fractalkine.
METHODS:
We transfected 293FT cells (a human kidney cell line) and B6/scl-7 cells with a rat CX3CL1-OFPSpark (C-terminal tag) expression plasmid (Sinobiologicals) to express membrane-bound fractalkine. Primary rat microglia were co-cultured with transfected 293FT cells, B6/scl-7 cells, or treated with soluble rat fractalkine (Peprotech).
RESULTS:
293FT cells were successfully transfected using Fugene 6®, but B6/scl-7 cells were not. Alternatively, B6/scl-7 cells were electroporated using Neon® Transfection System, but resulted in cell death. Microglia plated with FKN-expressing 293FT cells interacted favorably with the transfected cells, while those plated on untransfected 293FT cells exhibited unfavorable interactions and were rounded.
CONCLUSION:
These findings suggest transfecting B6/scl-7 cells with another method such as Lipofectamine 3000®. These models interact favorably with microglia compared to untransfected controls, showing these are viable models. Once stable transfections are achieved for both cell lines, these models will be used to: visualize cell-cell interactions of microglia in response to fractalkine signaling, quantify these interactions with Boyden chamber migration assays and CyQuant GF Fluorescent dye, and observe the regulation of NFκB in the signaling pathway of fractalkine.