Fluorescence‐activated sorting of fixed nuclei: a general method for studying nuclei from specific cell populations that preserves post‐translational modifications

Abstract

Long-lasting brain alterations that underlie learning and memory are triggered by synaptic activity. How activity can exert long-lasting effects on neurons is a major question in neuroscience. Signalling pathways from cytoplasm to nucleus and the resulting changes in transcription and epigenetic modifications are particularly relevant in this context. However, a major difficulty in their study comes from the cellular heterogeneity of brain tissue. A promising approach is to directly purify identified nuclei. Using mouse striatum we have developed a rapid and efficient method for isolating cell type-specific nuclei from fixed adult brain (fluorescence-activated sorting of fixed nuclei; FAST-FIN). Animals are quickly perfused with a formaldehyde fixative that stops enzymatic reactions and maintains the tissue in the state it was at the time of death, including nuclear localisation of soluble proteins such as GFP and differences in nuclear size between cell types. Tissue is subsequently dissociated with a Dounce homogeniser and nuclei prepared by centrifugation in an iodixanol density gradient. The purified fixed nuclei can then be immunostained with specific antibodies and analysed or sorted by flow cytometry. Simple criteria allow distinction of neurons and non-neuronal cells. Immunolabelling and transgenic mice that express fluorescent proteins can be used to identify specific cell populations, and the nuclei from these populations can be efficiently isolated, even rare cell types such as parvalbumin-expressing interneurons. FAST-FIN allows the preservation and study of dynamic and labile post-translational protein modifications. It should be applicable to other tissues and species, and allow study of DNA and its modifications.

Cell type-specific histone acetylation profiling of Alzheimer’s Disease subjects and integration with genetics

“…For each sample, we used FANS to collect NeuN+, Pu.1+, and NeuN-/Pu.1-nuclei to obtain putative neuronal, microglial, and other glial populations, respectively (Figure 1a, Supplementary Figure 2) 39 . On each collected population, we performed ChIP-Seq for H3K27ac, which is associated with transcriptionally active promoters and enhancers 40 .…”

“…We address these issues by profiling individual cell types deregulated during AD. We utilize fluorescence-activated nuclei sorting (FANS) 39 to purify neuronal, microglial and other glial populations in the dorsolateral prefrontal cortex (dlPFC) and hippocampus of subjects with and without AD pathology. Then, we perform chromatin immunoprecipitation and sequencing (ChIP-seq) for H3K27ac, which is associated with active promoters and enhancers 40 , to mark putative regulatory elements (peaks) in these populations.…”

Protein characterization of intracellular target-sorted, formalin-fixed cell subpopulations

“…In the current study, we established and validated a novel fixation/sorting/protein extraction method in which cell subpopulations can be identified and collected based on an intracellular marker and their protein expression characterized without bias from the larger population. By fixing cells immediately after tissue dissociation, representative cellular heterogeneity was maintained, and respective molecular expression was preserved 22 33 . This process allowed maintenance of cell characteristics outside original physiological environments without invoking potential changes due to standard 2D cell culturing conditions 7 19 .…”

Freedom of expression: cell‐type‐specific gene profiling

“…FACS‐based approaches to sorting nuclei exist in numerous organisms including mouse, Arabidopsis , and C. elegans . One notable addition to the repertoire is the batch isolate tissue‐specific chromatin for immunoprecipitation (BiTS‐ChIP) technique in Drosophila .…”