2. Desiccator (Fisher Scientific, Hampton, NH, USA)—Used to
remove air bubbles from the PDMS mixture.
3. Convection oven—Used for thermal curing of the PDMS
mixture.
2.3 Stamp Coating
1. Fibronectin (Sigma-Aldrich, St. Louis, MO, USA)—A 25 μg/
mL fibronectin in PBS solution is used to stamp the cell-culture
dish to promote cell adhesion.
2. Pluronic F127 (Sigma-Aldrich)—A 0.2% w/v solution of
Pluronic in DPBS is used to passivate surfaces and prevent
cell adhesion.
3. DPBS (Thermo Fisher Scientific)—DPBS is used to prepare
the fibronectin and Pluronic mixtures.
4. Ethanol—70% ethanol used to sterilize the PDMS stamp
surfaces.
5. 60-mm culture dishes and 35-mm Polystyrene dish (SigmaAldrich)—The dishes are used as the substrate for protein
fibronectin protein stamping and passivation with Pluronic.
6. Syringe filter (Millipore Sigma, Burlington, MA, USA)—A
0.22-μm syringe filter is used to filter the Pluronic solution
prior to application.
7. Syringes (Becton Dickinson, Franklin Lakes, NJ, USA)—3 mL
syringes used for the Pluronic passivation.
2.4 Cell Culture
1. Human Embryonic Kidney Cells (HEK293)—HEK293 cells
were grown in Dulbecco’s Modified Eagle Medium (DMEM)
(Thermo Fisher Scientific, Waltham, MA) containing 10% fetal
bovine serum (FBS) (Thermo Fisher Scientific) and 1% penicillin–streptomycin (10,000 U/mL) (Thermo Fisher
Scientific).
2. Trypsin—0.25% trypsin–EDTA solution (Thermo Fisher Scientific) is used for dissociating the cells.
2.5 Transfection
Using NFP-E
1. Nanofountain probe electroporation (NFP-E) system (Infinitesimal, Skokie, IL, USA)—NFP-E is used to deliver molecules
for transfection via localized electroporation.
2. Plasmid DNA—For transfection of HEK293 cells, a 4.2 kb
plasmid pSELECT-GFPzeo-mcs (Invitrogen, Carlsbad, CA)
is used to induce GFP expression and resistance to the antibiotic zeocin.
3. DPBS—The plasmid DNA stock solution is diluted in DPBS
(Thermo Fisher Scientific). A typical final concentration of
50 ng/μL of plasmid DNA is used for the transfections.
Nanofountain Probe Electroporation for Monoclonal Cell Line Generation
61
remove air bubbles from the PDMS mixture.
3. Convection oven—Used for thermal curing of the PDMS
mixture.
2.3 Stamp Coating
1. Fibronectin (Sigma-Aldrich, St. Louis, MO, USA)—A 25 μg/
mL fibronectin in PBS solution is used to stamp the cell-culture
dish to promote cell adhesion.
2. Pluronic F127 (Sigma-Aldrich)—A 0.2% w/v solution of
Pluronic in DPBS is used to passivate surfaces and prevent
cell adhesion.
3. DPBS (Thermo Fisher Scientific)—DPBS is used to prepare
the fibronectin and Pluronic mixtures.
4. Ethanol—70% ethanol used to sterilize the PDMS stamp
surfaces.
5. 60-mm culture dishes and 35-mm Polystyrene dish (SigmaAldrich)—The dishes are used as the substrate for protein
fibronectin protein stamping and passivation with Pluronic.
6. Syringe filter (Millipore Sigma, Burlington, MA, USA)—A
0.22-μm syringe filter is used to filter the Pluronic solution
prior to application.
7. Syringes (Becton Dickinson, Franklin Lakes, NJ, USA)—3 mL
syringes used for the Pluronic passivation.
2.4 Cell Culture
1. Human Embryonic Kidney Cells (HEK293)—HEK293 cells
were grown in Dulbecco’s Modified Eagle Medium (DMEM)
(Thermo Fisher Scientific, Waltham, MA) containing 10% fetal
bovine serum (FBS) (Thermo Fisher Scientific) and 1% penicillin–streptomycin (10,000 U/mL) (Thermo Fisher
Scientific).
2. Trypsin—0.25% trypsin–EDTA solution (Thermo Fisher Scientific) is used for dissociating the cells.
2.5 Transfection
Using NFP-E
1. Nanofountain probe electroporation (NFP-E) system (Infinitesimal, Skokie, IL, USA)—NFP-E is used to deliver molecules
for transfection via localized electroporation.
2. Plasmid DNA—For transfection of HEK293 cells, a 4.2 kb
plasmid pSELECT-GFPzeo-mcs (Invitrogen, Carlsbad, CA)
is used to induce GFP expression and resistance to the antibiotic zeocin.
3. DPBS—The plasmid DNA stock solution is diluted in DPBS
(Thermo Fisher Scientific). A typical final concentration of
50 ng/μL of plasmid DNA is used for the transfections.
Nanofountain Probe Electroporation for Monoclonal Cell Line Generation
61