228
HEK293 (ATCC), PC12 cells (PC12 Tet-Off, Clontech), any
other heterologous cell line, or primary cells that are amenable to
efficient transfection and allow studying protein–protein interactions (see Note 4).
1. Medium, depending on the cell line chosen (e.g., for HEK293
cells: DMEM 4.5 g glucose/l without l-glutamine, each with
and without phenol red). See complete media formulations for
HEK293 and PC12 cells below.
2. Fetal bovine serum (FBS), heat-inactivated, 500 ml (Life
Technologies)
3. Horse serum (HS), heat-inactivated, 500 ml (Life Technologies)
(only needed for PC12 cells).
4. GlutaMAX, 100×, 100 ml (Life Technologies).
5. Penicillin and Streptomycin, 100×, 100 ml (Life Technologies).
6. Opti-MEM (Life Technologies).
7. Poly-l-lysine, mol wt 70,000–150,000, diluted in a final concentration of 0.02 mg/ml in H 2 O (Sigma).
8. Lipofectamine 2000 transfection reagent (Life Technologies).
9. Passive lysis buffer, 5× (Promega, E1941).
10. Dual-Luciferase® Reporter 1000 Assay System (Promega,
E1980) (for an alternative using self-made substrates, see
Note 14).
1. 96-well plates: white with flat bottom for luciferase-based
assays (Falcon).
2. 96-well plates: clear with flat bottom for optical transfection
control and fluorescence-based assays (Falcon).
3. 75 cm
2
flasks (Falcon) or 15 cm dish (Falcon) (for maintenance of cell lines).
4. Cell culture facility including flow hood and incubator set to
5% CO 2 and 37 °C.
5. Luciferase reader (e.g., Mithras, Berthold Technologies or
Envision, PerkinElmer).
6. Fluorescence microscope (to check for transfection efficiency).
Prepare for each cell line used the appropriate media for maintenance and assay conditions.
1. HEK293 cells, assay medium: DMEM (4.5 g glucose/l, without l-glutamine and without phenol red), 500 ml, supplemented with 0.5% FBS and 2 mM GlutaMAX.
2. HEK293 cells, medium for maintenance: DMEM (4.5 g
glucose/l, without L-glutamine, with phenol red), 500 ml,
2.2 Cells
2.3 Reagents
2.4 Equipment
2.5 Media
Formulation
Jan P. Wintgens et al.
HEK293 (ATCC), PC12 cells (PC12 Tet-Off, Clontech), any
other heterologous cell line, or primary cells that are amenable to
efficient transfection and allow studying protein–protein interactions (see Note 4).
1. Medium, depending on the cell line chosen (e.g., for HEK293
cells: DMEM 4.5 g glucose/l without l-glutamine, each with
and without phenol red). See complete media formulations for
HEK293 and PC12 cells below.
2. Fetal bovine serum (FBS), heat-inactivated, 500 ml (Life
Technologies)
3. Horse serum (HS), heat-inactivated, 500 ml (Life Technologies)
(only needed for PC12 cells).
4. GlutaMAX, 100×, 100 ml (Life Technologies).
5. Penicillin and Streptomycin, 100×, 100 ml (Life Technologies).
6. Opti-MEM (Life Technologies).
7. Poly-l-lysine, mol wt 70,000–150,000, diluted in a final concentration of 0.02 mg/ml in H 2 O (Sigma).
8. Lipofectamine 2000 transfection reagent (Life Technologies).
9. Passive lysis buffer, 5× (Promega, E1941).
10. Dual-Luciferase® Reporter 1000 Assay System (Promega,
E1980) (for an alternative using self-made substrates, see
Note 14).
1. 96-well plates: white with flat bottom for luciferase-based
assays (Falcon).
2. 96-well plates: clear with flat bottom for optical transfection
control and fluorescence-based assays (Falcon).
3. 75 cm
2
flasks (Falcon) or 15 cm dish (Falcon) (for maintenance of cell lines).
4. Cell culture facility including flow hood and incubator set to
5% CO 2 and 37 °C.
5. Luciferase reader (e.g., Mithras, Berthold Technologies or
Envision, PerkinElmer).
6. Fluorescence microscope (to check for transfection efficiency).
Prepare for each cell line used the appropriate media for maintenance and assay conditions.
1. HEK293 cells, assay medium: DMEM (4.5 g glucose/l, without l-glutamine and without phenol red), 500 ml, supplemented with 0.5% FBS and 2 mM GlutaMAX.
2. HEK293 cells, medium for maintenance: DMEM (4.5 g
glucose/l, without L-glutamine, with phenol red), 500 ml,
2.2 Cells
2.3 Reagents
2.4 Equipment
2.5 Media
Formulation
Jan P. Wintgens et al.