3 Methods
3.1 Preparation of
Cells and Transfection
Day 1
1. Seed 600,000 HeLa cells in a 35 mm dish with a final volume of
2 mL of DMEM + 10% FBS + 1% Pen/Strep and keep it at
37
C with 5% CO 2 .
Day 2
2. Remove the medium from the dish and add 2 mL of fresh
prewarmed DMEM + 10% FBS without Pen/Strep.
3. Dilute 1 μg of the plasmid encoding the MCP-GFP-RBP
fusion protein in 250 μL of Opti-MEM. Simultaneously, dilute
10 μL of Lipofectamine 2000 in 250 μL of Opti-MEM (see
Note 3).
4. After 5 min, mix the two solutions and incubate at room
temperature for 25 min.
5. Subsequently, add the transfection mixture to the cells in a
dropwise manner.
6. Incubate the cells at 37
C with 5% CO 2 for 6 h.
7. Split the cells using standard cell culture techniques, seed
350,000 cells in a new 35 mm dish, and place it at 37
C with
5% CO 2 .
3.2 Seeding of Cells
on the CYTOO
Coverslips for Imaging
Day 4
1. Coating the CYTOO chip with fibronectin:
(a) Place the CYTOO chip in a 35 mm dish such that the
micropattern surface is facing upwards.
(b) Thaw 2 mL of 40 μg/mL fibronectin and add to the
35 mm dish containing the CYTOO chip. Additionally,
add 2 mL of PBS. Make sure that the chip is fully
immersed in the solution.
(c) Incubate the dish for 2 h at room temperature.
(d) Wash the chip by adding 4 mL of PBS to the existing
solution and then by removing 4 mL of the final solution.
Repeat this step three times and place the chip with PBS in
the sterile CYTOO chamber.
2. Seeding cells on the CYTOO chip:
(a) Prepare a HeLa cell suspension at a density of 60,000
cells/mL by trypsinization of the transfected cells and
filter them using a 40 μm cell strainer to ensure single
cells.
(b) Seed 2 mL of cell solution in the CYTOO chamber (after
removing PBS) (see Note 4).
Tethering Assay for RNA Mobility
273
3.1 Preparation of
Cells and Transfection
Day 1
1. Seed 600,000 HeLa cells in a 35 mm dish with a final volume of
2 mL of DMEM + 10% FBS + 1% Pen/Strep and keep it at
37
C with 5% CO 2 .
Day 2
2. Remove the medium from the dish and add 2 mL of fresh
prewarmed DMEM + 10% FBS without Pen/Strep.
3. Dilute 1 μg of the plasmid encoding the MCP-GFP-RBP
fusion protein in 250 μL of Opti-MEM. Simultaneously, dilute
10 μL of Lipofectamine 2000 in 250 μL of Opti-MEM (see
Note 3).
4. After 5 min, mix the two solutions and incubate at room
temperature for 25 min.
5. Subsequently, add the transfection mixture to the cells in a
dropwise manner.
6. Incubate the cells at 37
C with 5% CO 2 for 6 h.
7. Split the cells using standard cell culture techniques, seed
350,000 cells in a new 35 mm dish, and place it at 37
C with
5% CO 2 .
3.2 Seeding of Cells
on the CYTOO
Coverslips for Imaging
Day 4
1. Coating the CYTOO chip with fibronectin:
(a) Place the CYTOO chip in a 35 mm dish such that the
micropattern surface is facing upwards.
(b) Thaw 2 mL of 40 μg/mL fibronectin and add to the
35 mm dish containing the CYTOO chip. Additionally,
add 2 mL of PBS. Make sure that the chip is fully
immersed in the solution.
(c) Incubate the dish for 2 h at room temperature.
(d) Wash the chip by adding 4 mL of PBS to the existing
solution and then by removing 4 mL of the final solution.
Repeat this step three times and place the chip with PBS in
the sterile CYTOO chamber.
2. Seeding cells on the CYTOO chip:
(a) Prepare a HeLa cell suspension at a density of 60,000
cells/mL by trypsinization of the transfected cells and
filter them using a 40 μm cell strainer to ensure single
cells.
(b) Seed 2 mL of cell solution in the CYTOO chamber (after
removing PBS) (see Note 4).
Tethering Assay for RNA Mobility
273