2.4 Cell Culture
1. EA.hy925 cell line (passage 10–20) cultured in T25 flasks.
2. 5 mL Eppendorf tubes.
3. Round petri-dish (culture area 8.8 cm
2 ).
4. 70% ethanol.
5. Phosphate-buffered saline (PBS).
6. Trypsin.
7. Cell culture medium: Dulbecco’s modified Eagle’s medium
(DMEM) mixed with 10% fetal bovine serum (FBS) and 1%
penicillin-streptomycin.
2.5 Live cell Imaging
1. Live cell imaging chamber (temperature and CO 2 control).
2. Confocal microscope.
2.6 Immunostaining
1. Fixation solution: 4% paraformaldehyde in PBS.
2. Buffer solution: 0.05% Tween-20 in PBS.
3. 0.1% Triton X-100 solution in PBS.
4. Blocking solution: 1% BSA in PBS.
5. Phalloidin.
6. Hoechst (Invitrogen).
3 Methods
Carry out all procedures at room temperature, unless stated
otherwise.
3.1 LEP
1. Assemble the LEP configuration as shown in Fig. 1.
2. Switch on the stage. Open the LabVIEW program which controls the x-y stage movement. Set the stage to move at
100 mm/s in a zig-zag pattern (acceleration 1000 mm/s
2 ).
Test the stage movement without any loading.
3. Load the polymer solution into the syringe. Firmly attach the
metallic tip onto the syringe (see Note 1).
4. Place the syringe in the pump. Set the flow rate of 200 μL/h to
match the stage speed. Place the pump in the position where
the metallic tip points vertically toward the stage (see Note 2).
5. Attach one crocodile clip onto the metallic tip. The other
crocodile clip (ground) is attached to the stage.
6. Place the collecting substrate onto the stage. The selection of
the substrate material depends on the purpose of the experiment. For example, PDMS films and glass coverslips are often
used for cell assays. Place two initiators on either side of the
collecting substrate, leaving a 0.5 mm gap between the
130
Zhaoying Li et al.
1. EA.hy925 cell line (passage 10–20) cultured in T25 flasks.
2. 5 mL Eppendorf tubes.
3. Round petri-dish (culture area 8.8 cm
2 ).
4. 70% ethanol.
5. Phosphate-buffered saline (PBS).
6. Trypsin.
7. Cell culture medium: Dulbecco’s modified Eagle’s medium
(DMEM) mixed with 10% fetal bovine serum (FBS) and 1%
penicillin-streptomycin.
2.5 Live cell Imaging
1. Live cell imaging chamber (temperature and CO 2 control).
2. Confocal microscope.
2.6 Immunostaining
1. Fixation solution: 4% paraformaldehyde in PBS.
2. Buffer solution: 0.05% Tween-20 in PBS.
3. 0.1% Triton X-100 solution in PBS.
4. Blocking solution: 1% BSA in PBS.
5. Phalloidin.
6. Hoechst (Invitrogen).
3 Methods
Carry out all procedures at room temperature, unless stated
otherwise.
3.1 LEP
1. Assemble the LEP configuration as shown in Fig. 1.
2. Switch on the stage. Open the LabVIEW program which controls the x-y stage movement. Set the stage to move at
100 mm/s in a zig-zag pattern (acceleration 1000 mm/s
2 ).
Test the stage movement without any loading.
3. Load the polymer solution into the syringe. Firmly attach the
metallic tip onto the syringe (see Note 1).
4. Place the syringe in the pump. Set the flow rate of 200 μL/h to
match the stage speed. Place the pump in the position where
the metallic tip points vertically toward the stage (see Note 2).
5. Attach one crocodile clip onto the metallic tip. The other
crocodile clip (ground) is attached to the stage.
6. Place the collecting substrate onto the stage. The selection of
the substrate material depends on the purpose of the experiment. For example, PDMS films and glass coverslips are often
used for cell assays. Place two initiators on either side of the
collecting substrate, leaving a 0.5 mm gap between the
130
Zhaoying Li et al.