Preparation of Drosophila S2 cells for Light Microscopy

Summary

Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.

Abstract

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos¹, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.

S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media². In addition, they grow optimally and quickly at 21-24°C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.

Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)³. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Protocol

1. Preparing S2 cells for microscopy

Schneider S2 cells were derived from trypsinized late embryos of Oregon R Drosophila. Schneider's original culture consisted of a mixture of cell types but became more homogeneous with continued passage¹. They have been described as being macrophage-like (they are phagocytic) with hemocyte-like gene expression. S2 cells can be obtained from the ATCC, DGRC, or Invitrogen.

A. Selection of growth medium

A number of media have been used to culture S2 cells. None of the media mentioned below require a 5% CO₂ atmosphere.

....

Discussion

For the cell biology field, an ideal system would be inexpensive to maintain, easy to manipulate, and amenable to a variety of techniques. Drosophila S2 cells satisfy these requirements, and so they have rapidly become the system of choice for a growing number of cell biology labs.

We have presented a brief overview of the methods to prepare S2 cells for microscopy. A notable virtue of S2 cells is the fact that they grow well in normal atmosphere and room temperature; therefore, the.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
S2 cells	ATCC	CRL-1963	http://www.atcc.org/
S2 cells	DGRC	stock number 6	https://dgrc.cgb.indiana.edu/
S2 cells	Invitrogen	R690-07
Sf900 II	Invitrogen	10902-096	serum-free medium
HyClone SFX-Insect	Thermo Fisher Scientific, Inc.	SH30278	serum-free medium
Insect-Xpress	BioWhittaker	12-730	serum-free medium
Schneider’s S2 medium	Invitrogen	11720-034	Add 10% (final) heat-inactivated FBS to make complete medium.
Fetal bovine serum	Invitrogen	10438026	heat inactivated
100x antibiotic solution	MP Biomedicals	91674049	contains penicillin (10000 U/mL), streptomycin (10000 μg/mL), and amphotericin B (25 μg/mL)
Concanavalin A	MP Biomedicals	195283
Formaldehyde	Electron Microscopy Sciences	15714	32% solution
Methanol	Mallinckrodt Baker Inc.	9049	anhydrous, ACS grade
Molecular sieves	Acros Organics	19724	type 3A