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.
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.
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.
B. Culture conditions
S2 cells are easily maintained under normal lab temperature and atmosphere conditions. When imaging live S2 cells for extended times, the primary causes of cell death are dehydration and photo-toxicity. Dehydration is easily prevented by keeping sufficient medium in the dish on the microscope. Photo-toxicity is a trickier problem that requires minimizing the cells’ cumulative exposure to high-intensity, high-energy light while imaging frequently enough to capture interesting events with adequate spatial and temporal resolution.
2. Microscopy of S2 cells
A. Live cell microscopy
We use inverted microscopes to visualize live S2 cells plated on glass-bottom dishes. The glass-bottom dishes described below will keep the cells in a condition very similar to their normal culturing condition, and also allows live cells to be examined through microscopy-grade glass.
B. Fixed cell microscopy
3. Limitations/problems
As with any experimental system, there are limitations to the use of S2 cells. First, cultured S2 cells typically exhibit a low mitotic index (approximately 1% in serum-free media), whereas the mitotic indices for many transformed mammalian cell lines are as much as 10 fold higher. Therefore, for studies of mitotic phenotypes, S2 cell cultures require a longer search to find appropriately-staged cells. Second, for cell cycle studies, drug treatments are very effective at arresting S2 cells during specific cell cycle phases. However, compounds that have reversible cell-cycle arresting effects in cultured mammalian cells do not easily washout in S2 cells. Thus, protocols for synchronizing proliferating S2 cells have not been established. Third, S2 cells are not migratory nor do they display epithelial characteristics.
4. Expected Results
The biggest selling point for S2 cells is their utility as a model system. They are particularly useful for assessing the cellular functions of your protein of interest: S2 cells are treated by RNAi using dsRNA that is easily (and relatively cheaply) synthesized in the lab, and they serve well for live cell analysis and for immunofluorescence of fixed cells. In addition, a key benefit to S2 cells is the ease with which they can be maintained in the lab, using relatively inexpensive serum-free medium and no tissue culture incubator.
A typical experiment would involve plating S2 cells in an appropriate tissue culture container (say, a 6 or 96 well plate), adding home-made dsRNA to the wells, and then maintaining the cultures as the targeted proteins are gradually depleted by RNAi. Depending on the target proteins being depleted, the cells may show a morphological and/or behavioral change as a result of target protein knock-down. The time required to reduce target protein expression to a minimum is specific to the protein and should be determined by Western blotting.
Normally S2 cells do not adhere tightly to plastic or glass, and this would pose a problem for experiments requiring the microscopic examination of treated cells. However, S2 cells can be induced to flatten extensively by simply plating them on a surface coated with the lectin, concanavalin A (ConA). Within an hour of plating on ConA, most cells have lost their rounded appearance as a result of spreading extensively on the ConA surface (Figure 1). The cells are now ready for further processing (e.g., by fixation and immunostaining) or microscopic observation of live cells.
S2 cells can also be transfected (using a variety of chemical reagents or electroporation) to either transiently express an exogenous gene or to stably incorporate the gene into the genome (thus creating a cell line that maintains the exogenous gene through repeated divisions). Transfections can serve many purposes, like expression of fluorescently tagged proteins that mark structures of interest in fixed or live cells (Figure 2), or that are used as bait in in vivo pull-down or immunoprecipitation experiments, etc.
The method of analysis depends, of course, on the biological question being addressed by the experiment. A common method of analysis of RNAi-treated S2 cells is immunofluorescence of fixed cells (Figure 3). For example, immunofluorescence is typically used with genome-wide screens of Drosophila gene libraries to rapidly identify proteins with interesting activities in vivo. The analysis can be made more elaborate by simultaneously depleting multiple target proteins in S2 cells in order to examine the potential functional interactions between the target proteins. And the analysis can be extended to live cells, to observe the effect of RNAi on dynamic processes. Again, S2 cells are particularly well-suited for this type of analysis because they can plated on ConA-coated glass-bottom dishes and visualized for many hours on an inverted microscope without the need of an environmental chamber.
Figure 1. Phase contrast images (20x magnification) of S2 cells after plating on a concanavalin A-coated glass-bottom dish. The numbers indicate the minutes after plating. Note that the cells become phase dark as they flatten on the coated cover slip. Cell flattening is apparent by 15 min (arrows) and is essentially complete by 60 min. Right panel: Magnified image of attached cells; their extended and flattened margins are clearly visible (arrowhead). Scale bar (for the four left panels), 20 μm.
Figure 2. S2 cells transiently transfected with a fusion construct consisting of nucleophosmin (a marker for the nucleus) fused to the fluorophore, eGFP (green). These cells were plated on a ConA-coated glass-bottomed dish, and then imaged with DIC and epifluorescence. Scale bar, 15 μm.
Figure 3. Fixed S2 cells immunostained for PLP (green; a centriole marker), microtubules (red), and Hoechst-stained (blue) for chromosomes. Before fixation and immunostaining, the cells were subjected to a 4-day treatment with either control RNAi or RNAi to knock-down Ncd, a kinesin-like protein that promotes spindle pole “focusing”4. Note the splayed spindle poles and disorganized spindles in the cells treated with Ncd RNAi. Scale bar, 2.5 μm.
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, they can left on the microscope for extended periods without any special maintenance requirements. Even though they are generally somewhat rounded and loosely adherent under normal culturing conditions, S2 cells can be induced to extensively flatten for imaging. S2 cells cultured on glass-bottom dishes that have been pre-coated with concanavalin A will attach and spread thinly on the cover slip, making them excellent microscopy specimens.
The authors have nothing to disclose.
This work was supported in part by the National Cancer Institute P30 CA23074, the American Cancer Society Institutional Research Grant 74-001-31, and the Univ. of Arizona GI SPORE (NCI/NIH CA9506O).
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
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 Scientific | 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 | 9049 | anhydrous, ACS grade | |
Molecular sieves | Acros Organics | 19724 | type 3A |