Flow cytometric sorting of neuronal and glial nuclei from central nervous system tissue.
Due to the complex cellular heterogeneity of the central nervous system (CNS), it is relatively difficult to reliably obtain molecular descriptions with cell-type specificity. In particular, comparative analysis of epigenetic regulation or molecular profiles is hampered by the lack of adequate methodology for selective purification of defined cell populations from CNS tissue. Here, we developed a direct purification strategy of neural nuclei from CNS tissue based on fluorescence-activated cell sorting (FACS). We successfully fractionated nuclei from complex tissues such as brain, spinal cord, liver, kidney, and skeletal muscle extruded mechanically or chemically, and fractionated nuclei were structurally maintained and contained nucleoproteins and nuclear DNA/RNA. We collected sufficient numbers of nuclei from neurons and oligodendrocytes using FACS with immunolabeling for nucleoproteins or from genetically labeled transgenic mice. In addition, the use of Fab fragments isolated from papain antibody digests, which effectively enriched the specialized cell populations, significantly enhanced the immunolabeling efficacy. This methodology can be applied to a wide variety of heterogeneous tissues and is crucial for understanding the cell-specific information about chromatin dynamics, nucleoproteins, protein-DNA/RNA interactions, and transcriptomes retained in the nucleus, such as non-coding RNAs.
Okada
Seiji
S
Superstar Program Stem Cell Unit, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan. seokada@ortho.med.kyushu-u.ac.jp
Saiwai
Hirokazu
H
Kumamaru
Hiromi
H
Kubota
Kensuke
K
Harada
Akihito
A
Yamaguchi
Masahiro
M
Iwamoto
Yukihide
Y
Ohkawa
Yasuyuki
Y
D000818:Animals; D002467:Cell Nucleus / Q000648:ultrastructure*; D002469:Cell Separation / Q000379:methods*; D002490:Central Nervous System / Q000033:anatomy & histology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009457:Neuroglia / Q000166:cytology*; D009474:Neurons / Q000166:cytology*