Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

Summary

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Abstract

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis^1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)³ under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver⁴ or the control of the pan-da neuron-specific GAL4^21-7 driver⁵. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Protocol

General Comments on Magnetic Bead Sorting of Drosophila Peripheral Neurons (Total Timing for the Completion of the Protocol: 2.5-3 hours) Standard lab procedures for maintaining a clean, RNAse free environment must be observed at all times to prevent RNA degradation. When the Drosophila larval cuticle is dissected and placed in the cell dissociation buffer, the peripheral neurons are one of the last cells to detach from the cuticle. We have exploit…

Discussion

The protocol presented here is optimized for the isolation and purification of peripheral neurons which adhere tightly to the inner surface of the Drosophila third instar larval cuticle using a magnetic bead cell sorting strategy. While we have used this protocol to specifically isolate Drosophila da neurons, applications of this protocol to the isolation of other cell types that adhere to the cuticle in larval or pupal stages of development (e.g. epithelia, muscle, other peripheral neurons) can be adapted by varying a …

Materials

Material Name	Type	Company	Catalogue Number	Comment
10X Phosphate Buffered Saline (PBS)		MP Bioproducts	PBS10X02	Diluted to 1X working solution
10X Liberase Blendzyme 3		Roche	11814176001	Diluted to 1X working solution (28 Wünsch units/vial)
RNase-AWAY		Sigma-Aldrich	83931
Biotinylated Rat anti-Mouse-CD8a antibody		Invitrogen	MCD0815	100 μg/ml stock concentration
BSA (Bovine Serum Albumin), Fraction V		GibcoBRL	11018-017	Prepare a 1% BSA solution in PBS
Dynabeads M-280 Streptavidin		Invitrogen	11205D	1 μl can bind 0.05-0.10 μg of biotinylated antibody
PicoPure RNA Isolation Kit		Molecular Devices	KIT0204	Follow manufacturer’s instructions

Equipment

• (10) Neodymium block magnets (K & J Magnetics, Inc., Cat. #B444B), alternatively DynaMag™-2 (Invitrogen, Cat. #123-21D) may be used.
• Kontes Glass Tissue Grinder, 2 ml working capacity, with large clearance pestle (Cat. #885300-0002)
• Cell filters, e.g. MACS Pre-Separation Filter (Miltenyi Biotec, Cat. #130-041-407)
• 35 mm petri-dishes coated with Sylgard (Dow Corning Corporation, Cat. #3097358-1004)
• Dissecting tools: two pairs of Dumont No. 5 forceps (Fine Science Tools, Cat. #11251-20)
• Vannas spring micro-dissection scissors (Fine Science Tools, Cat. #15000-08)
• Pipettes (P-1000, P-100, P-10) with disposable tips
• Standard hemocytometer to count the cells and estimate purity
• Pasteur pipettes with fire polished tips of standard diameter, narrowed to ~50% of standard diameter and narrowed to ~25% of standard diameter for trituration
• Table top micro-centrifuge (1-16,000 (x) g)
• Vortex
• Fluorescent stereo-microscope (a Leica MZ16FA was used in this protocol)