Fluorescence-activated sorting of fixed nuclei: a general method for studying nuclei from specific cell populations that preserves post-translational modifications.
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.
Inserm UMR-S 839, Paris, France; Université Pierre & Marie Curie, Sorbonne Universités, Paris, France; Institut du Fer à Moulin, Paris, France.
Lucile
L
Marion-Poll
Enrica
E
Montalban
Annie
A
Munier
Denis
D
Hervé
Jean-Antoine
JA
Girault
D000818:Animals; D001921:Brain / Q000166:cytology; D002458:Cell Fractionation / Q000379:methods; D002467:Cell Nucleus / Q000145:classification / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D006657:Histones / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009928:Organ Specificity; D011499:Protein Processing, Post-Translational
D006657:Histones
epigenetics; flow cytometry; fluorescence-activated cell sorting; formaldehyde fixation; histone modifications; nuclear purification