title: Neurocytometry: Flow Cytometric Sorting of Specific Neuronal Populations from Human and Rodent Brain.
abstract: Flow cytometry has the potential to facilitate understanding of the heterogeneous responses of diverse brain cell populations to a variety of stimuli. However, existing methods of applying flow cytometry to brain tissues are each limited in certain ways. They either require genetically labeled cells to achieve separation of specific populations, are not applicable to previously fixed tissue, or are not compatible with downstream mRNA analysis. Here, we describe a group of related methods that overcome many previous limitations and allow robust sorting and downstream molecular analysis of highly enriched populations of specific neuronal and non-neuronal cells from any mammalian brain. We illustrate these techniques, which are compatible with antibodies for both nuclear and non-nuclear epitopes and do not require transgenic animals, with three examples. First, we describe the separation and downstream mRNA analysis of four types of cortical interneurons (somatostatin, parvalbumin, calretinin, and calbindin) from paraformaldehyde-fixed rat brain sections. Second, we demonstrate separation of neurons and non-neurons from zinc-fixed mouse brain cortical sections followed by analysis of enzymatic activity (ACE2 activity) and mRNA expression. Third, we show that routinely fixed post-mortem human brain can be analyzed by isolating parvalbumin-containing neurons from cortical samples that were fixed for periods of up to 8 weeks in formalin. In each case, sorted cell identity was confirmed with mRNA analysis. The neurocytometry methodology described here has the potential to significantly expand studies to analyze the effects of drugs, environmental manipulations, and disease states on the nucleic acid and protein content of specific brain cell populations.
authors.affiliation: Department of Pharmacology and Experimental Therapeutics, LSU Health Sciences Center , New Orleans, Louisiana 70112, United States.
authors.forename: David
authors.initials: D
authors.lastname: Martin
authors.affiliation: Department of Pharmacology and Experimental Therapeutics, LSU Health Sciences Center , New Orleans, Louisiana 70112, United States.
authors.forename: Jiaxi
authors.initials: J
authors.lastname: Xu
authors.affiliation: Department of Pharmacology and Experimental Therapeutics, LSU Health Sciences Center , New Orleans, Louisiana 70112, United States.
authors.forename: Connie
authors.initials: C
authors.lastname: Porretta
authors.affiliation: Department of Pharmacology and Experimental Therapeutics, LSU Health Sciences Center , New Orleans, Louisiana 70112, United States.
authors.forename: Charles D
authors.identifier: 0000-0002-0615-0646
authors.initials: CD
authors.lastname: Nichols
mesh_terms: D000085962:Angiotensin-Converting Enzyme 2; D000806:Angiotensin-Converting Enzyme Inhibitors; D000818:Animals; D001921:Brain / Q000166:cytology*; D064032:Calbindin 2 / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D010455:Peptides; D007703:Peptidyl-Dipeptidase A / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D016704:Synapsins / Q000235:genetics / Q000378:metabolism
chemical_list: D000806:Angiotensin-Converting Enzyme Inhibitors; D064032:Calbindin 2; C556151:DX600 peptide; D009419:Nerve Tissue Proteins; D010455:Peptides; D012333:RNA, Messenger; D016704:Synapsins; D007703:Peptidyl-Dipeptidase A; C000705307:ACE2 protein, human; C000705328:Ace2 protein, mouse; C000705329:Ace2 protein, rat; D000085962:Angiotensin-Converting Enzyme 2
keywords: FACS; brain; flow cytometry; interneuron; neurocytometry; neuron