Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Summary

Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing.

Abstract

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological conditions. Post-mitotic neurons represent a major cell type affected by aging. Although multiple mammalian models of neuronal aging exist, they are challenging and expensive to establish. The roundworm Caenorhabditis elegans is a powerful model to study neuronal aging, as these animals have short lifespan, an available robust genetic toolbox, and well-cataloged nervous system. The method presented herein allows for seamless isolation of specific cells based on the expression of a transgenic green fluorescent protein (GFP). Transgenic animal lines expressing GFP under distinct, cell type-specific promoters are digested to remove the outer cuticle and gently mechanically disrupted to produce slurry containing various cell types. The cells of interest are then separated from non-target cells through fluorescence-activated cell sorting, or by anti-GFP-coupled magnetic beads. The isolated cells can then be cultured for a limited time or immediately used for cell-specific ex vivo analyses such as transcriptional analysis by real time quantitative PCR. Thus, this protocol allows for rapid and robust analysis of cell-specific responses within different neuronal populations in C. elegans.

Introduction

Over the past several decades, the metazoan model organism Caenorhabditis elegans has been a tremendous asset in the study of neurons, neuronal circuitry and its role in physiological and behavioral responses, and aging-associated neurodegenerative diseases. A unique feature of C. elegans is that the animals are transparent, allowing for the lineage of all adult somatic cells to be mapped¹. C. elegans also harbors manageable quantity of neurons, leading to the morphology and connectivity of the nervous system being well understood². The invariant cell lineage, short lifespan, and abundance of h....

Protocol

1. Preparation and collection of aged worms for cell isolation

NOTE: Explained below is the isolation of cholinergic neurons from the transgenic unc-17::GFP strain (OH13083) obtained from the Caenorhabditis Genetics Center (CGC) strain repository at the University of Minnesota. It is imperative to maintain sterile conditions to prevent contamination from fungi or bacteria.

1. Prepare and synchronize worms via the bleaching method as described by T. Stiernagle

Discussion

The roundworm C. elegans is a well-established and powerful model to study neuronal health and disease². With ample genetic tools to manipulate these animals and manageable quantity of precisely mapped various neuron types, a great deal of data can be collected with a relatively small quantity of material. Here, we outline an optimized method to isolate distinct neurons from whole animals. By disrupting the worm’s outer cuticle proteins, a slurry of various cell types can be isolate.......

Materials

Name	Company	Catalog Number	Comments
6-well plate	Fisher Scientific	12-556-004
Agar, Molecular Biology Grade	VWR	A0930
CaCl2	Sigma	C5670
Chloroform	Sigma	496189
Contess Automated Cell Counter	Invitrogen	Z359629
DTT	USBiological	D8070
Ethanol	Decon Labs	2701
FBS	Omega Scientific	FB-02
Fluorodeoxyuridine	Sigma	F0503
HEPES	Sigma	H3375
Isopropanol	VWR	BDH1133
KCl	Amresco	O395
KH2PO4	USBiological	P5110
Kimwipes	Kimberly-Clark Professionals	7552
Leibovitz's L-15 Medium	Gibco	21083027
MgCl2	Sigma	M8266
MgSO4	USBiological	M2090
Na2HPO4·7H2O	USBiological	S5199
NaCl	VWR	X190
NaOCl (Bleach)	Clorox
NaOH	Amresco	O583
Penicillin-Streptomicen	Fisher Scientific	15140122
Peptone Y	USBiological	P3306
Pronase E	Sigma	7433	protease mixture from Streptomyces griseus
SDS	Amresco	O227
SMT1-FLQC fluorescence stereomicroscope	Tritech Research
Sucrose	USBiological	S8010
SuperScript IV One-Step synthesis kit	ThermoFisher	12594025
TRIzol	Invitrogen	15596026	phenol and guanidine isothiocyanate solution
Trypan Blue Stain	Invitrogen	T10Z82
α-GFP magnetic beads	MBL	D153-11