Primary Cell Cultures from Drosophila Gastrula Embryos

Summary

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Abstract

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Protocol

1. Preparation of fly cages

1. Grow Drosophila (wild-type strains such as Oregon-R or any genotype) in convenient containers such as 32-oz insect cups.
2. Transfer newly eclosed flies to large embryo-collection cages or fly population cages.
3. Move the cages into a room with a temperature of ~25 °C and humidity ~65% in a light 12-h and a dark 12-h cycle to facilitate the collection of synchronous embryos.
4. Maintain the flies in the cages by feeding them killed yeast paste streaked on molasses plates. Change the molasses plates (with killed yeast paste) once a day for 2-3 d.

2. Collection of synchro....

Discussion

The developmental pattern of primary cultures of Drosophila cells can vary greatly, possibly due to the several variables during the primary cell preparation process. First of all, flies used for egg laying should be young (within a week old) and healthy (free of viral or bacterial infection). Unfertilized or infected embryos must be avoided, as they are useless and cells derived from those embryos cannot differentiate and will only survive for 1-2 days. Second, during the homogenization process, it is import.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Shields and Sang M3 medium	Sigma-Aldrich	S3652
Fetal bovine serum	JRH Biosciences	12103-500M
Insulin from bovine pancreas	Sigma-Aldrich	I-6634
Large embryo collection cages	Genesee Scientific	59-101
#25 US standard testing sieve	VWR international	57334-454
#45 US standard testing sieve	VWR international	57334-462
#120 US standard testing sieve	VWR international	57334-474
Dounce tissue grinder, Wheaton 7 mL	VWR international	62400-620
Dounce tissue grinder,Kontes 40 mL	VWR international	KT885300-0040
Dounce tissue grinder,Kontes 40 mL	VWR international	KT885300-0100
Costar, 384-well plate	VWR international	29444-078
Lab-Tek II Chambered coverglass	Fisher Scientific	171080