Imaging Orc1 cellular dynamics in Drosophila S2 cells

S2 Transient Transfection

Commercial Reagents and Materials

• Tissue culture treated 6-well plates

• Complete ESF921 (Prepare with ESF921 (Expression Systems #96-001-01) supplemented to 1% FBS and 1x antibiotic/antiomycotic (Gibco #15240-062))

• Effectene (Invitrogen #301425) (Includes Buffer EC, Enhancer, and Effectene, see below)

• 35 mm glass bottom imaging dish (such as CellVis #D35-20-1.5-N)

User provided Reagents

• Gene of interest inserted into an insect cell suitable expression plasmid

Protocol:

Day1 - 

A. Seed a 6-well plate with 3 mL of S2 cells at a density of 1e6 cells/mL in Complete ESF921

B. Incubate cells 24 hours at 27 degrees Celsius

Day 2 - 

A. Prepare a 200 ng/uL stock of plasmid DNA in ddH2O

B. Add 2 uL of 200 ng/uL plasmid to 96 uL of Buffer EC and flick tube to mix

C. Add 6.4 uL of Enhancer to DNA/Buffer EC mixture and vortex to mix

D. Incubate the sample for 5 minutes at room temperature

E. Centrifuge the sample briefly to collect solution in bottom of tube

F. Add 10 uL of Effectene to each transfection mixture and vortex 10 seconds to mix

G. Incubate the sample for 10 minutes at room temperature

H. During incubation, gently aspirate medium from cells in 6-well plate

I. Immediately and gently add 1.6 mL of Complete ESF921 to each well

J. After incubation, add 600 uL of Complete ESF921 to each transfection mixture and quickly mix by pipetting twice up-and-down, and immediately add the solution dropwise to cells in 6-well plate

K. Mix the plate by gently swirling in a “figure 8” motion and incubate the cells for 16 hours at 27 degrees Celsius

Day 3 - 

A. Very gently aspirate medium and replace with 2.5 mL of Complete ESF921

B. Incubate an additional 8-24 hours before downstream analysis (optimal time post-transfection for downstream analysis should be determined empirically) 

Day 3-4 - 

A. Assess results with technique of interest (i.e. western blot analysis, confocal imaging, pull down, etc)

        A'. If imaging, resuspend cells with the medium from the well and transfer cells to a glass bottom tissue culture dish before imaging

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