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MethodsX. 2015 Jan 15;2:39-46. doi: 10.1016/j.mex.2015.01.002. eCollection 2015.

Quick and reliable method for retina dissociation and separation of rod photoreceptor perikarya from adult mice.

Author information

1
Department of Biology II, Center for Integrated Protein Science Munich (CIPSM), Ludwig Maximilians University Munich, Grosshadernerstrasse 2, 82152 Planegg-Martinsried, Germany ; Department of Medical Biology, Medical University-Plovdiv, Blvd. Vasil Aprilov 15A, 4000 Plovdiv, Bulgaria.
2
Center for Integrated Protein Science Munich CIPSM at the Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377 München, Germany.
3
Department of Biology II, Center for Integrated Protein Science Munich (CIPSM), Ludwig Maximilians University Munich, Grosshadernerstrasse 2, 82152 Planegg-Martinsried, Germany.

Abstract

A pure and abundant population of adult rod perikarya can be exploited in different studies concerning nuclear functions such as gene expression analyses which aim at elucidating the relationship between cell type and disease [1]. Sorting is based either on specific cell-surface markers or fluorescently labeled reporter proteins. Here, we describe a simple and reliable method for separation of rod photoreceptor perikarya without the use of staining procedures or transgenic mice. This method is limited, however, to sorting rod photoreceptors from adult mouse retina. Mature rods possess an inverted nuclear architecture which is determined by the optical functions of these nuclei [2]. The high backscatter of heterochromatin in the core of the nucleus can be used as a selection criterion for FAC-sorting by forward and sideward scatter. •The procedure for retina dissociation using the Papain Dissociation System (Wothington Biochemical Corporation) was optimized.•An easy to follow step-by-step protocol for retina dissociation was devised.•Rod perikarya were FAC-sorted by forward and sideward scatter based solely on the high backscatter of heterochromatin in their nuclei.

KEYWORDS:

FACS; Mouse retina; Papain dissociation; Retina isolation; Rod cell perikarya; Sorting of mouse rod photoreceptorsn

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