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
      ....

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