Year: 2017
Type: article
Abstract: THE common usage of animal models in a variety of preclinical studies is supported by appropriate species-specific antibodies to be utilized in immunohistochemistry (IHC), western blotting, and flow cytometry (FC) assays.Other than the technical advantages (sophisticated surgical manipulations due to their size), modest cost (relative to larger animals), and the standardized results, the similarities in metabolic activity and physiology of neurological disorders to humans make rats appropriate for neurological disease or disorders models (1,2).However, rat-based assays are not as comprehensive or standardized as mouse or human based assays are, partially because there is a shortage in rat-specific antibodies.Rat-specific antibodies are now becoming commercially available, which allows us to set standardize criteria for rat origin cells of interest.As our research focus is in neurotherapy, we are interested specifically in microglial cells, which are the innate immune cells in the brain (3) and spinal cord (4).Microglial cells play a critical role in traumatic brain injuries (TBI) and spinal cord injuries (SCI), and their presence, activation, and effect are highly investigated in those models (4-6).Microglial characterization via FC would save many hours of work as a substitute for IHC analysis, yield unbiased statistics, and overall help research move at a faster pace (7).Here we present multicolor phenotyping panels for assessing microglia derived from rat brain or spinal cord for their activation states, polarization, and number (see Table 1).The microglial cells used are immediately isolated from fresh brain or spinal cord tissues, using a Neural Tissue Dissociation kit, followed by myelin removal and purification using anti-rat CD11b/ c microbeads. BACKGROUNDThe Optimized Multicolor Immunofluorescence Panel (OMIP) was designed to phenotypically characterize rat-derived microglial cells isolated from brain or spinal cord, in TBI or SCI rat models.Central nervous system (CNS) injury immediately results in a robust inflammatory cell-mediated response.The key players in the response to the injury are microglial cells, which are a major cellular component of the CNS innate immune system.In normal situations, microglia maintains a dynamic and ramified phenotype, while an injury results in a dramatic amoeboid phenotype change.CNS injuries also lead to infiltration of macrophages to the injury site and both microglia and macrophages share the expression of surface markers such as CD45, CD11b/c, and CX 3 CR 1 .The common way of identifying microglia
Source: Cytometry Part A
DOI: 10.1002/cyto.a.23267
Language: English
Subfield: Neurology
Field: Neuroscience
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W2762034969