Transfektion von Schneider Zellen (S2)

For transfection, I either use the lipofectiopn (e.g. with FuGene) especially when I just want to have protein overexpression, that is using pAct5-GAL4 (or pMT-GAL4) with any pUAST-xx. However, in case I want to transfect several plasmids, e.g. co-overexpression of Crb, Sdt, Lin7 and DPATJ; I use the CaPO4 protocol, because I get a moderate but more robust overexpression. In general the CaPO4 is more robust, if the pH of your transfection reagent is adjusted properly.

Independent of the transfection method, I start with 1 x 10(6) cells / 6-well-slot (35 mm diameter). The transfection ratio depends on that the cells are growing nicely (logarithmically growing cells). To reach this, I passage them 1:5 (1:7.5) every 5 days. The best is, if the cells adhere directly after seeding and then start to form clumps, looking like grapes.

For lipofection I typically use 0.75 µg pAct5GAL4 and 0.75 µg of pUAST-xx, dilute with 100 µl medium w/o serum, add 3 µl lipofection reagaent, thus a 1:2 ratio. Furthermore, Schneider cells are slow, thus harvesting cells after 72 h instead of after 48 h (post transfection) can be beneficial.

In general, I used fly extracts to help the cells but meanwhile found out that I can do without and still get about 50% transfection (measured as GFP positive cells in FACS). What helps is to feed the cells directly before adding the transfection reagent, thereby also getting rid of the non-adherent cells (which you always have when seeding 1 x10(6). Also, always pre-warm the medium, Schneider cells do not like cold medium - but do not store the medium at 25¡C, it is sensitive. Along this line, Schneider cells definitely need heat-inactivated serum. Concerning medium, we used the Gibco Schneider cell medium, the Sigma medium and also M3+BPYE; without any obvious differences. However, if there are precipitates in the medium, the cells may not adhere due to that calcium is precipitating and thereby lowering the concentration the cells require. Vice versa, non-adherent Schneider cells can easily be forced to adhere by adding CaCl2.

General transient transfection

reference:

items required:

• cells in logarithmic phase, not from confluent cultures
• plasmid DNA (1 µg/µl) and/or carrier DNA (either self made chromosomal DNA [needs shearing] or commercially available hering sperm DNA [salmon sperm DNA]; concentration: 1 µg/µl), column purified or CsCl-gradient
• commercially available transfection reagent, e.g. FuGENE HD transfection reagent or similar product

procedure:

day 1:

day 2:

1. start with DNA (either 2 ?g plasmid DNA or at least 1 ?g plasmid DNA supplemented with 1 ?g carrier DNA)
2. dilute DNA with 100 µl serum-free cell culture medium or dH2O
3. add transfection reagent, here: add 3 µl FuGENE HD transfection reagent
4. mix and incubate for 15 min (for S2 cells: 30 min), do not spin !
5. add transfection complex to cells (either drop-wise or below the surface of the medium)

day 3:

day 4: