Purification of Low-abundant Cells in the Drosophila Visual System

Jing Peng; Ivan J. Santiago; Matthew Y. Pecot

doi:10.3791/58474

Purification of Low-abundant Cells in the Drosophila Visual System

JoVE Journal
Genetics

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JoVE Journal Genetics

Purification of Low-abundant Cells in the Drosophila Visual System

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07:09 min

September 26, 2018

DOI: 10.3791/58474-v

Jing Peng¹, Ivan J. Santiago¹, Matthew Y. Pecot¹

¹Department of Neurobiology,Harvard Medical School

Overview

This article presents a cell dissociation protocol for isolating low-abundance cells in the Drosophila visual system using fluorescence activated cell sorting (FACS). This method aids in understanding the organization and generation of diverse cell types.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Genomics

Background

Understanding cell diversity is crucial in biology.
Cell sorting techniques are essential for transcriptomic analysis.
Drosophila serves as a model organism for studying visual systems.
Low-abundance cells pose challenges in isolation and analysis.

Purpose of Study

To develop a protocol for efficient cell dissociation.
To facilitate the study of low-abundance cell types.
To enhance transcriptomic and genomic analyses in Drosophila.

Methods Used

Fluorescence activated cell sorting (FACS).
Preparation of papain solution for cell dissociation.
Use of Complete Schneider's Medium (CSM).
Incubation and mixing techniques for optimal enzyme activity.

Main Results

Successful isolation of low-abundance cells.
Improved efficiency in cell purification for analysis.
Protocol enables detailed study of cell types in the visual system.
Facilitates further research into cellular organization and function.

Conclusions

The developed protocol is effective for isolating specific cell types.
FACS can significantly enhance genomic studies.
This method contributes to understanding complex biological questions.

Frequently Asked Questions

What is the main advantage of this protocol?

It allows for efficient purification of low-abundance cells in the Drosophila visual system.

How does this method contribute to biological research?

It helps address questions about cell type diversity and organization.

What is FACS?

Fluorescence activated cell sorting is a technique used to sort cells based on their fluorescence characteristics.

What is papain used for in this protocol?

Papain is used to dissociate cells for easier isolation.

Why is Drosophila a good model organism?

Drosophila offers a simple and well-characterized visual system for studying cellular processes.

What is Complete Schneider's Medium?

CSM is a nutrient medium used to support cell growth and maintenance.

Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting (FACS).

This method can help address key questions in biology, such as how diverse cell types are generated and organized into higher order structures with specific functions. The main advantage of this technique is that it facilitates efficient purification of cells present at a low abundance in the Drosophila visual system for transcriptomic and genomic analysis. On Tuesday of week six of the protocol, 45 minutes before the dissections, retrieve one vial of papain powder from four degrees Celsius and incubate it at room temperature.

Next, set aside Complete Schneider's Medium, or CSM, in an Eppendorf tube at room temperature to add to the papain powder later. Five to ten minutes before the dissections, use a syringe to add the room temperature CSM to the vial of papain directly through the cap to a concentration of 100 units per milliliter. To mix, first invert the vial to capture any powder stuck on the inside of the cap and then pipette the contents up and down.

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