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OMIP-041

Optimized multicolor immunofluorescence panel rat microglial staining protocol

Naama E. Toledano Furman, Karthik S. Prabhakara, Supinder Bedi, Charles S. Cox Jr, Scott D. Olson Cytometry PART A, Volume 93, Issue 2, 182–185 (2017)

PURPOSE: 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.

CELL TYPE: Primary Microglia Cells

MACHINE: BD LSR2

Marker Target Clone Fluorochrome Purpose
Dead cells Rat N/A Ghost Dye Violet 510 APC-Cy7
CD45 Rat OX-1 APC-Cy7 PE-Cy7
CD11b/c Rat OX-42 PE-Cy7 BV421
P2RY12 Rat polyclonal Brilliant Violet 421 PE
CD32 Rat D34-485 PE APC
CD86 Rat APC PE
CD200 R Rat OX-102 PE Alexa Fluor 647
RT1B Rat OX-6 Alexa Fluor 647 PerCP Cy5.5
CD163 Rat ED2 PerCP-Cy5.5 V510