Abstract
Aim: In the field of neuroinflammation, identifying specific effects of pharmacological agents and other factors is problematic given the relative difficulty and expense in obtaining and culturing primary microglia. Immortalized microglial cell lines are very useful, but only a limited number have been characterized for inflammatory signaling. Therefore, characterization of lipopolysaccharide (LPS)-induced toll-like receptor 4 (TLR4) signaling in CHME-5, a microglial cell line, is expected to be of value as an experimental model of inflammatory signaling in the central nervous system (CNS).
Methods: It was recently suggested that CHME-5 cells are of rat origin, not human, hence, verification of this claim using short tandem repeat genotype sequencing, along with immunoblotting, reverse transcription-polymerase chain reaction, and immunocytochemistry techniques to validate that CHME-5 retain morphological, phenotypical, and functional characteristics of primary microglia were undertaken.
Results: LPS induced inhibitor kappa B-alpha and nuclear factor-kappa B (NF-κB) p65 activation, NF-κB p65 binding activity, and tumor necrosis factor alpha gene expression. Additionally, results also confirmed the maintenance of microglial phenotype as seen with increased cluster of differentiation 68 gene and protein expression, immunofluorescence, and the absence of glial fibrillary acidic protein-immunoreactivity. TLR4 gene expression and immunofluorescence were significantly increased after LPS treatment.
Conclusion: These data demonstrate that CHME-5 cells are not human, but are indeed a beneficial tool for studying microglial inflammatory signaling.
Methods: It was recently suggested that CHME-5 cells are of rat origin, not human, hence, verification of this claim using short tandem repeat genotype sequencing, along with immunoblotting, reverse transcription-polymerase chain reaction, and immunocytochemistry techniques to validate that CHME-5 retain morphological, phenotypical, and functional characteristics of primary microglia were undertaken.
Results: LPS induced inhibitor kappa B-alpha and nuclear factor-kappa B (NF-κB) p65 activation, NF-κB p65 binding activity, and tumor necrosis factor alpha gene expression. Additionally, results also confirmed the maintenance of microglial phenotype as seen with increased cluster of differentiation 68 gene and protein expression, immunofluorescence, and the absence of glial fibrillary acidic protein-immunoreactivity. TLR4 gene expression and immunofluorescence were significantly increased after LPS treatment.
Conclusion: These data demonstrate that CHME-5 cells are not human, but are indeed a beneficial tool for studying microglial inflammatory signaling.
| Original language | American English |
|---|---|
| Pages (from-to) | 219 |
| Number of pages | 231 |
| Journal | Neuroimmunology Neuroinflammation |
| Volume | 4 |
| State | Published - 19 Oct 2017 |