230
positions on the clear plate to guarantee an optical control of
transfection efficiency (see Note 8).
7. Prepare the transfection mix. Dilute the Lipofectamine 2000
150-fold in Opti-MEM supplemented with 1% GlutaMAX,
vortex for 1–2 s, and incubate for 5 min. For each 96-well, we
use 0.2 μl Lipofectamine 2000 that is diluted in 30 μl OptiMEM. We suggest preparing a 7× master mix for all conditions
to be tested.
8. Pipette the Lipofectamine/Opti-MEM mix onto the DNA,
mix (vortex), and incubate for 20 min at room temperature.
9. Remove medium from each well entirely (be careful not to
remove the cells) and transfer the DNA/transfection mix (30
μl per well) onto the cells.
10. After 1 h, add 60 μl medium for maintenance without
antibiotics.
11. Depending on whether a constitutive or stimulus-dependent
protein–protein interaction assay is run,
●
●
proceed to step 12 for constitutive, or
●
●
proceed to step 13 for agonist and agonist/antagonist
assays.
●
●
Common experimental paradigms are schematically displayed in Fig. 4.
12. Constitutive assay: Incubate for 12–24 h. Typically, we incubate constitutive assays for 20 h. Before lysing the cells, verify
the transfection efficiency using the clear optical control plate.
By this time, the transfected plasmids should express considerably, and transfection efficiency, as determined by GFP signals,
should be at least 30–50% to obtain stable assay conditions,
otherwise start optimizing transfection parameters (see Note 9).
●
●
Proceed to step 17.
13. Agonist and agonist/antagonist assays: Incubate overnight.
14. On the next day, verify transfection efficiency (c.f. step 12, see
Note 9) and replace the medium with 80 μl assay medium to
induce the starvation of cells.
15. After 16–24 h, add appropriate stimuli in 80 μl assay medium,
with the agonists in double concentration. If studying agonist/antagonist effects, add the antagonists 1 h before the agonists (see Note 10).
16. Incubate for 6–24 h, depending on the assay setup. This step
strongly depends on the individual interaction measured, the
cell type used, or biological question addressed. For a first
approach, we incubate for 18–20 h (see Note 11).
17. Aspirate the medium and add 30 μl of 1x Passive Lysis Buffer
to each well. Incubate the 96-well plate on a horizontal rocking platform (100–150 rpm) for 10 min at room temperature.
Jan P. Wintgens et al.
positions on the clear plate to guarantee an optical control of
transfection efficiency (see Note 8).
7. Prepare the transfection mix. Dilute the Lipofectamine 2000
150-fold in Opti-MEM supplemented with 1% GlutaMAX,
vortex for 1–2 s, and incubate for 5 min. For each 96-well, we
use 0.2 μl Lipofectamine 2000 that is diluted in 30 μl OptiMEM. We suggest preparing a 7× master mix for all conditions
to be tested.
8. Pipette the Lipofectamine/Opti-MEM mix onto the DNA,
mix (vortex), and incubate for 20 min at room temperature.
9. Remove medium from each well entirely (be careful not to
remove the cells) and transfer the DNA/transfection mix (30
μl per well) onto the cells.
10. After 1 h, add 60 μl medium for maintenance without
antibiotics.
11. Depending on whether a constitutive or stimulus-dependent
protein–protein interaction assay is run,
●
●
proceed to step 12 for constitutive, or
●
●
proceed to step 13 for agonist and agonist/antagonist
assays.
●
●
Common experimental paradigms are schematically displayed in Fig. 4.
12. Constitutive assay: Incubate for 12–24 h. Typically, we incubate constitutive assays for 20 h. Before lysing the cells, verify
the transfection efficiency using the clear optical control plate.
By this time, the transfected plasmids should express considerably, and transfection efficiency, as determined by GFP signals,
should be at least 30–50% to obtain stable assay conditions,
otherwise start optimizing transfection parameters (see Note 9).
●
●
Proceed to step 17.
13. Agonist and agonist/antagonist assays: Incubate overnight.
14. On the next day, verify transfection efficiency (c.f. step 12, see
Note 9) and replace the medium with 80 μl assay medium to
induce the starvation of cells.
15. After 16–24 h, add appropriate stimuli in 80 μl assay medium,
with the agonists in double concentration. If studying agonist/antagonist effects, add the antagonists 1 h before the agonists (see Note 10).
16. Incubate for 6–24 h, depending on the assay setup. This step
strongly depends on the individual interaction measured, the
cell type used, or biological question addressed. For a first
approach, we incubate for 18–20 h (see Note 11).
17. Aspirate the medium and add 30 μl of 1x Passive Lysis Buffer
to each well. Incubate the 96-well plate on a horizontal rocking platform (100–150 rpm) for 10 min at room temperature.
Jan P. Wintgens et al.