Transfection S2 cells

Transfect S2 cells with an appropriate expression construct (refer to discussion in Cherbas and Cherbas, this volume). 

For antibody staining, place 50-100 ll of transfected S2 cells in each well of a multiwell microscope slide. 

S2 cell staining. Screen only the positive clones from the dot blots on transfected S2 cells attached to multiwell slides. If there were too many positives to screen, then select the strongest ones. Using the multiwell slides, it is reasonable to screen 250 or... 

Procedures for transfection by calcium phosphate-DNA coprecipitation and by electroporation are given in our earlier reviews (Cherbas et al. 1994 Cherbas and Cherbas 1998). The electroporation protocol for Kc cells (Cherbas et al. 1994) can be modified for S2 cells by increasing the voltage from 440 V/cm (Kc) to 715 V/cm (S2) (Cherbas and Cherbas 1998) the higher voltage also works well for S3 cells, for the shibire line EH34A3, and for the haploid line D (K. Klueg and M.A.T. Muskavitch, pers. comm.). 

To generate arrays of plasmid, transfect cells by calcium phosphate-DNA coprecipitation, using 1 ml of precipitate for 3 ml of cells. The precipitate contains 20 xg of supercoiled plasmid DNA/ml, a mixture of the plasmid of interest, and a plasmid carrying a selectable marker. For S2 cells, if the composition of the plasmids does not lead to selection against their presence, incorporation of approximately 1000 plasmid copies per haploid genome will result, in the form of arrays containing the same ratio of plasmids that was present in the input mix. The products of this transformation procedure in other lines cells are less completely characterized. 

Test No. 2 Staining of Transfected Drosophila S2 Cultured Cells, 399 Test No. 3 Staining of Drosophila Tissues—Helpful Hints, 400 Protocol 21.3 Dot Blot, 401 Protocol 21.4 Drosophila S2 Cell Staining, 404 MONOCLONAL ANTIBODIES, 406... 

Cotransfection with a GPP construct is recommended to control for transfection and to ascertain that the cells detected by the antiserum are actually of the transfected population. Thus, if the antiserum recognizes the correct overexpressed Drosophila antigen, bright staining should be detected in only a small fraction of the cells, and these same cells should also express the GFP construct. Even if the protein is endogenously expressed in S2 cells, there should stUl be an obvious difference in protein level between endogenous and overexpressed. 

Test No. 2 Staining of Transfected Drosophila S2 Cultured Cells... 

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