Подготовка Drosophila S2 клеток для световой микроскопии
Summary
Дрозофилы Шнайдер (S2) клетки становятся все более популярной системой для обнаружения и функционального анализа генов. Нашей целью является описание некоторых из микроскопических методов, которые делают S2 клетках, таких все более важной экспериментальной системы.
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
Discussion
Для поля клеточной биологии, идеальная система была бы недорогой в обслуживании, легко манипулировать, и поддаются различных методов. Drosophila S2 клетках удовлетворяют этим требованиям, и поэтому они быстро стали системой для все большего числа клеточных биологии лаборатории.
<p class…Disclosures
The authors have nothing to disclose.
Acknowledgements
Эта работа была выполнена при частичной поддержке Национального института рака P30 CA23074, American Cancer Society институциональных исследований Грант 74-001-31, а Univ. Аризона И. SPORE (NCI / NIH CA9506O).
Materials
| 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 |
References
- Schneider, I. Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol. 27, 353-365 (1972).
- Rogers, S. L., Rogers, G. C. Culture of Drosophila S2 cells and their use for RNAi-mediated loss-of-function studies and immunofluorescence microscopy. Nat Protoc. 3, 606-611 (2008).
- Rogers, S. L., Rogers, G. C., Sharp, D. J., Vale, R. D. Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle. J Cell Biol. 158, 873-884 (2002).
- Goshima, G. Genes required for mitotic spindle assembly in Drosophila S2 cells. Science. 316, 417-421 (2007).
