Summary

की तैयारी ड्रोसोफिला S2 कोशिकाओं लाइट माइक्रोस्कोपी के लिए

Published: June 03, 2010
doi:

Summary

ड्रोसोफिला श्नाइडर कोशिकाओं (S2) और जीन की खोज और कार्यात्मक विश्लेषण के लिए एक तेजी से लोकप्रिय प्रणाली है. हमारा लक्ष्य को सूक्ष्म तकनीक है कि इस तरह के एक तेजी से महत्वपूर्ण प्रायोगिक प्रणाली S2 कोशिकाओं को बनाने के कुछ का वर्णन है.

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. माइक्रोस्कोपी के लिए S2 कोशिकाओं की तैयारी श्नाइडर S2 कोशिकाओं ओरेगन आर ड्रोसोफिला के trypsinized देर से भ्रूण से प्राप्त किए गए . श्नाइडर मूल संस्कृति सेल प्रकार का एक मिश्रण के शामिल है, लेकिन …

Discussion

कोशिका जीव विज्ञान के क्षेत्र के लिए एक आदर्श व्यवस्था बनाए रखने के लिए सस्ती हो, हेरफेर करने के लिए आसान होता है, और तकनीक की एक किस्म करने के लिए उत्तरदायी ड्रोसोफिला S2 कोशिकाओं इन आवश्यकताओं को सं…

Disclosures

The authors have nothing to disclose.

Acknowledgements

इस काम के हिस्से में राष्ट्रीय कैंसर संस्थान p30 CA23074, अमेरिकन कैंसर सोसायटी संस्थागत अनुसंधान 74-001-31 अनुदान, और Univ द्वारा समर्थित किया गया. एरिजोना सैनिक बीजाणु (NCI / एनआईएच 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

  1. Schneider, I. Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol. 27, 353-365 (1972).
  2. 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).
  3. 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).
  4. Goshima, G. Genes required for mitotic spindle assembly in Drosophila S2 cells. Science. 316, 417-421 (2007).
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Cite This Article
Buster, D. W., Nye, J., Klebba, J. E., Rogers, G. C. Preparation of Drosophila S2 cells for Light Microscopy. J. Vis. Exp. (40), e1982, doi:10.3791/1982 (2010).

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