Isolating Cell Populations from Brain Tissue Using Fluorescence-Activated Nuclei Sorting

Abstract

Source: Mussa, Z., et al. Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting. J. Vis. Exp. (2021)

This video demonstrates the procedure for isolating and sorting nuclei from fresh-frozen human brain tissue using fluorescence-activated nuclei sorting (FANS), which enables the study of different cell populations for various neurological applications.

Protocol

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

1. Buffer preparation

NOTE: If performing downstream RNA sequencing, thoroughly treat all workspaces and tools with RNase Decontamination Solution to prevent mRNA degradation.

1. Prepare lysis buffer.
   1. Dissolve the following in distilled water (H₂O) up to 50 mL: 5.47 g of sucrose (0.32 M final), 250 µL of 1 M calcium chloride (CaCl₂) (5 mM final), 150 µL of 1 M magnesium acetate (Mg(CH₃COO)₂)(3 mM final), 10 µL of 500 mM ethylenediaminetetraacetic acid (EDTA) (0.1 mM final), 500 µL of 1 M Tris-hydrochloric acid (Tris-HCl) (pH 8) (10 mM final), 50 µL of Triton X-100 (0.1% final), and 17 µL of 3 M dithiothreitol (DTT, 1 mM final, add fresh) (see the Table of Materials).
2. Prepare sucrose buffer
   1. Dissolve the following in distilled H₂O up to 50 mL: 30.78 g of sucrose (1.8 M final), 150 µL of 1 M Mg(CH₃COO)₂ (3 mM final), 500 µL of 1 M Tris-HCl (pH 8) (10 mM final), 17 µL of 3 M DTT (1 mM final, add fresh).

2. Frozen tissue dissociation into single-nucleus suspension

1. Add 4 mL of ice-cold lysis buffer to a 7 mL glass tissue douncer (grinder), and keep on ice. Dissect approximately 200-400 mg of the fresh-frozen human adult cortex, dounce approximately 50x, and transfer the tissue homogenate to a 12 mL ultracentrifuge polypropylene tube.
2. Using a 5 mL pipette, add 6.5 mL of ice-cold sucrose buffer to the bottom of the ultracentrifuge tube, taking care not to disturb the layer between sucrose and the tissue homogenate.
   ​NOTE: If processing additional samples, carefully balance the tubes by weight by adding additional lysis buffer to the lighter sample. Precise balancing of the ultracentrifuge tubes is essential to prevent damage to the rotor and ensure rotation at the proper speed.

3. Ultracentrifugation

1. Ultracentrifuge the lysate at 24,400 rpm (101,814 × g) for 1 h at 4 °C. Carefully aspirate the supernatant and debris without disturbing the pellet.
   NOTE: A pellet may not be visible if starting with less than 200 mg of tissue.
2. Add 600 µL of phosphate-buffered saline (PBS) (Calcium (Ca²⁺), Magnesium (Mg²⁺) free) to the nuclei pellet and incubate on ice for 10 min before resuspending.
3. Pipette up and down 50 times to resuspend the nuclei pellet on ice.
4. Use a hemocytometer to visualize the intact nuclei under a microscope and to ensure the concentration of the resuspension is above 10⁵ nuclei/mL before proceeding to the next step (combine 10 µL of the resuspended nuclei with 10 µL of trypan blue).

4. Antibody incubation

1. To immunotag the sample for fluorescence-activated nuclei sorting (FANS), add 500 µL of the resuspended sample pellet, 490 µL of 1x PBS (Ca²⁺, Mg²⁺ free), 10 µL of 10% bovine serum albumin (BSA) (0.1% final), 1 µL of mouse anti-NeuN antibody conjugated to AF(Alexa fluor)555 (NeuN-AF555, 1:1000 final concentration), and 1 µL of mouse anti-PAX6 antibody conjugated to allophycocyanin (PAX6-APC, 1:1000 final concentration).
   NOTE: Alternative antibodies conjugated to other fluorophores may be added. Here, mouse anti-OLIG2 conjugated to AF488 (1:1000 concentration) was used with favorable results.
2. Perform 4′,6-diamidino-2-phenylindole (DAPI)-only and single-color controls to set up gating parameters, using a small amount of the sample as necessary.
   1. To perform an AF555 single-color control, add 20 µL of the resuspended sample pellet, 970 µL of 1x PBS (Ca²⁺, Mg²⁺ free), 10 µL of 10% BSA (0.1% final), and 1 µL of NeuN-AF555 antibody (1:1000 concentration).
   2. To perform an APC single-color control, add 20 µL of the resuspended sample pellet, 970 µL of 1X PBS (Ca²⁺, Mg²⁺ free), 10 µL of 10% BSA (0.1% final), and 1 µL of PAX6-APC antibody (1:1000 concentration).
   3. To perform DAPI (4',6-diamidino-2-phenylindole)-only control, add 20 µL of the resuspended sample pellet, 970 µL of 1X PBS (Ca²⁺, Mg²⁺ free), and 10 µL of 10% BSA (0.1% final) (DAPI will be added later, see 4.4).
      NOTE: If more than two antibodies are used, performing fluorescence-minus-one (FMO) control is recommended in addition to the single-color controls.
3. Incubate the samples and the controls with rotation in the dark for 1 h at 4 °C.
4. Add DAPI at 1:1000 to all the samples and controls, and proceed with sorting.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10x PBS pH 7.2	Invitrogen	70013073
ANTI-NEUN ANTIBODY CLONE A60	Millipore	MAB377A5MI	mouse anti-NeuN conjugated to a fluorescent compound AF555 (excitation, 553 nm; emission, 568 nm)
ANTI-OLIG2 ANTIBODY CLONE 211	Millipore	MABN50A4MI	mouse anti-OLIG2 conjugated to a fluorescent compound AF488 (excitation, 499 nm; emission, 520 nm)
Bovine Serum Albumin	Fisher	BP9704-100
Bright-Line Counting Chamber	Hausser Scientific	3110V
Calcium Chloride Anhydrous	Fisher	C614-3
Cell Strainers, 40 µM	SP Scienceware	136800040
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)	Invitrogen	D1306
Dounce Tissue Grinder	WHEATON	357542
FACS Sorter	BD Biosciences		BD FACSAria III
Magnesium Acetate Tetrahydrate	Fisher	M13-500
PAX6 (PAX6/496) - 100 TESTS	Novus	NBP234705J
Sucrose, crystal certified, ACS, 500 mg	Fisher	S5500
SW 41 Ti Swinging-Bucket Rotor	Beckman Coulter	331362
Tris-HCl, 1M Solution, pH 8.0, Molecular Biology Grade, Ultrapure	Thermo Scientific	J22638AE
TritonX-100	Fisher	BP151-500	non-ionic surfactant in lysis buffer
Trypan Blue Solution, 0.4%	Gibco	15250061
Ultracentrifuge	Beckman Coulter Optima XE-100	A94516
Ultracentrifuge tubes PP 9/16 X 3-1/2	Beckman Coulter	331372
UltraPure Distilled Water (RNAse-, DNAse-free)	Invitrogen	10977023	referred to as distilled water
Ultrapure EDTA	Life Technologies	15576-028