Glycosylation in S2 cells is very much like that in SF9, Sf21, and High Five cells. The nature of Drosophila N-linked glycosylation is less complex than mammalian glycosylation – it is generally of the paucimannose type, and is not trimmed and sialylated. O-linked glycosylation is similar, although not identical, to mammalian cells. However, correct glycosylation profiles are difficult to obtain, also glycosylation from CHO and HEK293 differ from human glycosylation. Human O-glycosylation can be divided into N-acetylGalactosamine linked (mucin type), N-acetylGlucosamine lined (O-GlcNAc type) and xylose linked (proteoglycans) families. The most abundant form is the mucin type, while O-GlcNac has only been found for cytoplasmic and nuclear proteins, the proteoglycans are of less interest here. In CHO cells, O-glycosylation results in terminally sialated mucin type glycans, with a low percentage core-1 structure (T-antigen) reported. In Drosophila S2, cell O-glycosylation is less complex than in human or mammalian cells. Unlike human proteins, O-GlcNAc has been found on an external protein. It appears that O-GlcNAc can occur in specific cases and are linked to the regulation of the protein, in this case the Notch receptor. However, mucin type glycosylation is the dominant O-glycosylation type in S2 cells and has been shown to be of the Tn-antigen (GalNAcalpha-Ser/Thr) and the core-1 structure (T-antigen) (Galbeta1-3GalNAcalpha-Ser/Thr) forms.

Gentamycin

  • 10 µg/mL Inhibits bacterial protein synthesis.

Penicillin-Streptomycin

  • 100 U/mL Penicillin Inhibits bacterial cell wall synthesis.
  • 100 µg/mL Streptomycin Inhibits bacterial protein synthesis.

Amphotericin B

  • Fungizone 0.25 µg/mL Binds sterols and interferes with membrane permeability.

No, and in fact CO2 is toxic to S2 cells.

Humidification is only necessary for monolayer cultures in tissue culture plates. Plates can also be wrapped in parafilm and put in a box with a beaker with water. For other cultures, humidification is not necessary.

The accumulating dogma seems to indicate that CMV expresses in a number of cell types. For example, CMV drives expression in frog oocytes. The following articles discuss the CMV promoter in S2 cells. One provides evidence that it works well and one finds that it does not. Indicates that CMV promoter does not work in Kc1 and S2 cells: Gene 1997 Apr 1;188(2):183-90 Pfeifer TA, Hegedus DD, Grigliatti TA, Theilmann DA Baculovirus immediate-early promoter-mediated expression of the Zeocin resistance gene for use as a dominant selectable marker in dipteran and lepidopteran insect cell lines. Demonstrates that CMV promoter does work in Drosophila cells: Nucleic Acids Res 1987 Mar 11;15(5):2392 Sinclair JH The human cytomegalovirus immediate early gene promoter is a strong promoter in cultured Drosophila melanogaster cells.

Yes. D. Mel is short for Drosophila melanogaster, which is the proper species name for the cells commonly referred to as Schneider S2 cells or just S2 cells.

We recommend 110 rpm for shake flasks with or without baffles.

S2 cells can be grown at 23-27 °C but they will recover after excursions up to 37 °C. However, we routinely grow our cells at 25 °C.

Yes, Baculovirus can infect S2 cells, however the virus will not replicate in the infected cells and will not be able to infect subsequent cells. No high-titer stocks can be generated from S2 cells.

Some of the advantages are: a more homogeneous glycosylation profile; better reproducibility between manufacturing runs; several options for cultivation modes, such as perfusion that allows for long cultivation times with daily harvests; no cell lysis, resulting in lower levels of contaminating host cell proteins.

The baculovirus system is a lytic system (the cells lyse upon infection with a recombinant Baculovirus containing the gene of interest) while the S2 platform is stable insect cell expression. Insect cells are transfected with an expression vector, followed by selection and screening of polyclonal or monoclonal high-producers.

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