2. Plasmid expressing the chimeric fusion protein of MCP tandem dimer, 2x-GFP, and RBP (full-length/domain of interest). The domain of interest can be cloned at either the N- or
the C-terminus of the fusion protein (see Note 1).
3. Cell culture incubator.
4. Dulbecco’s modified Eagle medium (DMEM) supplemented
with 10% (v/v) Tet-free fetal bovine serum (FBS) and 1%
(v/v) penicillin and streptomycin (pen/strep).
5. DMEM supplemented with 10% (v/v) Tet-free FBS without
pen/strep.
6. Phosphate-buffered saline (PBS).
7. Trypsin-EDTA solution.
8. 35 mm Cell culture dish or 6-well plate.
9. CYTOO coverslips with cross-bow printed pattern (CYTOO
SA, CW-M-A X18, or CW-L-A X18).
10. CYTOO chamber (CYTOO SA, 30-010).
11. 40 μg/mL Fibronectin.
12. Automated cell counter and counting slides.
13. 1 mg/mL Doxycycline.
14. Lipofectamine 2000.
15. Opti-MEM.
16. 40 μm Cell strainer.
2.2 Imaging System
1. Wide-field or spinning disk confocal microscope (see Note 2).
2. 100 Â 1.45NA PlanApo TIRFM oil immersion objective.
3. Back-illuminated EMCCD camera.
4. Emission filter for GFP fluorescence.
5. Solid-state laser (100 mW 491 nm).
6. Motorized X, Y, Z-piezo controlled stage.
7. Incubation box around microscope providing heating and CO 2
regulation.
2.3 Image Analysis
1. Fiji software with TrackMate plug-in [21, 22].
2. Diff2fit [18].
3. HMM-Bayes [23].
4. All code and analysis tools are available from the Chao lab
(https://data.fmi.ch/PublicationSupplementRepo/?
group¼gchao).
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the C-terminus of the fusion protein (see Note 1).
3. Cell culture incubator.
4. Dulbecco’s modified Eagle medium (DMEM) supplemented
with 10% (v/v) Tet-free fetal bovine serum (FBS) and 1%
(v/v) penicillin and streptomycin (pen/strep).
5. DMEM supplemented with 10% (v/v) Tet-free FBS without
pen/strep.
6. Phosphate-buffered saline (PBS).
7. Trypsin-EDTA solution.
8. 35 mm Cell culture dish or 6-well plate.
9. CYTOO coverslips with cross-bow printed pattern (CYTOO
SA, CW-M-A X18, or CW-L-A X18).
10. CYTOO chamber (CYTOO SA, 30-010).
11. 40 μg/mL Fibronectin.
12. Automated cell counter and counting slides.
13. 1 mg/mL Doxycycline.
14. Lipofectamine 2000.
15. Opti-MEM.
16. 40 μm Cell strainer.
2.2 Imaging System
1. Wide-field or spinning disk confocal microscope (see Note 2).
2. 100 Â 1.45NA PlanApo TIRFM oil immersion objective.
3. Back-illuminated EMCCD camera.
4. Emission filter for GFP fluorescence.
5. Solid-state laser (100 mW 491 nm).
6. Motorized X, Y, Z-piezo controlled stage.
7. Incubation box around microscope providing heating and CO 2
regulation.
2.3 Image Analysis
1. Fiji software with TrackMate plug-in [21, 22].
2. Diff2fit [18].
3. HMM-Bayes [23].
4. All code and analysis tools are available from the Chao lab
(https://data.fmi.ch/PublicationSupplementRepo/?
group¼gchao).
272
Varun Bhaskar et al.