A Technique to Isolate Nuclei from Non-neuronal Cells in the Hippocampal Dentate Gyrus

Abstract

Source: Kerloch, T., et al. Fluorescence-Activated Nuclei Negative Sorting of Neurons Combined with Single Nuclei RNA Sequencing to Study the Hippocampal Neurogenic Niche. J. Vis. Exp. (2022).

The video demonstrates a method for isolating intact nuclei from glial and stem cells in the mouse hippocampal dentate gyrus (DG). The DG tissue is mechanically dissociated in a cold homogenization buffer to release the nuclei. Tissue debris and other released organelles are discarded, and the nuclei are stained with a DNA stain and a fluorophore-conjugated antibody specific for the neuronal nuclear antigen (NeuN). Fluorescence-activated nuclei sorting is then employed to isolate the NeuN-negative nuclei of non-neuronal cells.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines

1. Dissection of the DG (Timing: 15 min)

1. Prepare nuclei isolation media 1 and 2 (NIM1 and NIM2), homogenization buffer (HB), and wash media (WM) (Table 1). Place all the buffers, media, reagents, and tools on ice until needed. Put the Dounce homogenizer (see Table of Materials) on ice during preparation (minimum 1 h before the homogenization step).
   NOTE: NIM1 can be prepared and stored at 4 °C for up to 6 months. NIM2, HB, and WM should be....

Results

Figure 1: Preparation of a single nuclei suspension from the dissected DG of adult mice for snRNA-seq of non-neuronal populations. Flow diagram describing the main steps of the protocol that include dissection of mouse DG, preparation of a suspension of s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5ml microtube	Eppendorf	30124537
10.00µm Flouresbrite YG Carboxylate Microspheres	Polysciences	15700-10
15 mL polypropylene centrifuge tubes	Corning	430052
2 pairs of sterile Dumont #5 forceps	Fine Science Tools	11252-30
4′,6-diamidino-2-phenylindole (DAPI)	Sigma Aldrich	D9564-10MG
4150 TapeStation System	Agilent	N/A
5 mL round bottom high clarity polypropylene test tube with snap cap	Falcon	352063
5 mL round bottom polystyrene test tube with cell strainer snap cap	Falcon	352235
50 mL polypropylene centrifuge tubes	Corning	430829
70 µm cell strainer	Falcon	352350
8 peak SPHERO Rainbow Calibration Particles	BD Biosciences	RCP-30-5A
Accudrop Beads	BD Biosciences	N/A
Allegra X-30R Centrifuge	Beckman Coulter	N/A
Anti-NeuN antibody, clone A60, Alexa Fluor 488 conjugated	Millipore	MAB377X
BD FACSAria Fusion Flow Cytometer	BD Biosciences	N/A
Beckman Coulter MoFlo XDP	Beckman Coulter	N/A
BSA 7.5%	Gibco	15260037
Dithiothreitol (DTT)	Thermo Scientific	R0861
Dounce tissue grinder set: mortar, loose pestle (A) and tight pestle (B)	KIMBLE	D8938-1SET
Eppendorf Tubes Protein LoBind 1.5ml	Eppendorf	30108116
Halt, 100x Protease inhibitor	ThermoFisher	78429
KCl	Any chemical supplier		Laboratory made
LUNA-FX7 Automated Cell counter	Logos Biosystems	N/A
MgCl2	Any chemical supplier		Laboratory made
N°10 guarded sterile disposable scalpels	Swann-Morton	6601
Nuclease-free water	Sigma Aldrich	W4502-1L
Pair of sterile student surgical scissors	Fine Science Tools	91401-12
PBS	Any chemical supplier		Laboratory made
RNase Inhibitor 40 U µl-1	Ambion	AM2684
RNasin 40 U µl-1	Promega	N211A
Sterile Petri dish	Corning	430167
Sucrose	Sigma Aldrich	59378-500G
Tris buffer, pH 8.0	Any chemical supplier		Laboratory made
Triton X-100 10% (v/v)	Sigma Aldrich	T8787-250ML
Trypan blue	Invitrogen	T10282