10. Wait for 15 min to allow the transfer of the fibronectin from
the pillars onto the well.
11. In the meantime, take the pluronic solution prepared in step 7
and filter 1 mL per stamp using a 22 μm syringe filter.
12. Carefully remove the chip from the well with forceps and add
1 mL of the filtered pluronic solution (this solution will passivate the parts of the dish that do not contain fibronectin). Let
pluronic sit for 45 min.
13. Remove the pluronic solution with a pipette.
14. Add 25,000 cells per well in a total of 2 mL of culture media
(cell number can vary based on experimental needs and
cell type).
15. Place dish in incubator and transfect the cells that form colonies over the micropatterned regions the next day (see Note 1).
3.4 Cell Culture
on Stamped
Substrates
1. Harvest HEK293 cells by adding trypsin and collecting the
detached cells. Dissociate the clusters by gentle pipetting and
then count the cells.
2. Plate the cells on the micropatterned petri dishes by putting
5 Â 10
4 cells in 2 mL DMEM per dish and put them in the
incubator (37
C and 5% CO 2 ) overnight to allow them to
adhere.
3.5 Transfection
Using NFP-E (Fig. 2b)
1. Observe the cells on the micropatterned petri dish to confirm that they have adhered on the fibronectin stamped
regions to form isolated circular colonies. Wash the petri
dish with PBS to remove debris and unattached cells and
put fresh media.
2. Fill the NFP tip slowly with 20 μL of the plasmid solution using
an Eppendorf Microloader. Ensure that no bubbles are trapped
by visual inspection or under the microscope.
3. Clean the electrodes with ethanol.
4. Load the NFP tip on the piezo-actuated arm of the system,
which is mounted on the microscope column.
5. Place a petri dish with the patterned cells on the microscope
stage. Switch to phase contrast and bring the colonies of interest in focus (10Â or 20Â objective).
6. Lower the NFP tip using the controller until it touches the top
surface of the media. Bring the tip to the center of the field of
view and slowly lower the tip again until it is slightly above the
cells (~100 μm).
7. Calibrate the position of the tip using the NFP-E software.
8. Make sure that the resistance reading is stable (~30–50 MΩ)
(see Note 2).
Nanofountain Probe Electroporation for Monoclonal Cell Line Generation
65
the pillars onto the well.
11. In the meantime, take the pluronic solution prepared in step 7
and filter 1 mL per stamp using a 22 μm syringe filter.
12. Carefully remove the chip from the well with forceps and add
1 mL of the filtered pluronic solution (this solution will passivate the parts of the dish that do not contain fibronectin). Let
pluronic sit for 45 min.
13. Remove the pluronic solution with a pipette.
14. Add 25,000 cells per well in a total of 2 mL of culture media
(cell number can vary based on experimental needs and
cell type).
15. Place dish in incubator and transfect the cells that form colonies over the micropatterned regions the next day (see Note 1).
3.4 Cell Culture
on Stamped
Substrates
1. Harvest HEK293 cells by adding trypsin and collecting the
detached cells. Dissociate the clusters by gentle pipetting and
then count the cells.
2. Plate the cells on the micropatterned petri dishes by putting
5 Â 10
4 cells in 2 mL DMEM per dish and put them in the
incubator (37
C and 5% CO 2 ) overnight to allow them to
adhere.
3.5 Transfection
Using NFP-E (Fig. 2b)
1. Observe the cells on the micropatterned petri dish to confirm that they have adhered on the fibronectin stamped
regions to form isolated circular colonies. Wash the petri
dish with PBS to remove debris and unattached cells and
put fresh media.
2. Fill the NFP tip slowly with 20 μL of the plasmid solution using
an Eppendorf Microloader. Ensure that no bubbles are trapped
by visual inspection or under the microscope.
3. Clean the electrodes with ethanol.
4. Load the NFP tip on the piezo-actuated arm of the system,
which is mounted on the microscope column.
5. Place a petri dish with the patterned cells on the microscope
stage. Switch to phase contrast and bring the colonies of interest in focus (10Â or 20Â objective).
6. Lower the NFP tip using the controller until it touches the top
surface of the media. Bring the tip to the center of the field of
view and slowly lower the tip again until it is slightly above the
cells (~100 μm).
7. Calibrate the position of the tip using the NFP-E software.
8. Make sure that the resistance reading is stable (~30–50 MΩ)
(see Note 2).
Nanofountain Probe Electroporation for Monoclonal Cell Line Generation
65