4 Notes
1. The microcontact printing protocol may need to be optimized
if different matrix proteins and substrates are used than those
discussed here. The various substrates, proteins and stamp
polymers used in microcontact printing are discussed in the
following reference [14].
2. In some cases, the contact detection based on the resistance
change might not work due to the presence of air bubbles or
clogging of the probe tip by the transfectant. In such cases a
new pipette with fresh transfectant should be used. The biomolecule concentration can also be lowered to prevent
clogging.
3. If other cells are used, the transfection efficiency may not be
optimal. The pulse parameters and the biomolecule concentration may need to be adjusted to obtain maximum transfection
efficiency. Typically, the voltage amplitude (between 10 V and
25 V) and number of pulses (between 50 and 500) should be
changed to improve transfection.
4. The cell viability may be low in some experiments. This may
be because the pulse profile is too harsh for the cell type. The
voltage amplitude and number of pulses should be reduced in
this case. Keeping the cells for a long time outside the incubator and DNA toxicity due to high plasmid concentrations are
other causes of low viability. Typically, cells should not be kept
outside the incubator for more than 20 min, and lower plasmid concentrations (20 ng/μL to 200 ng/μL) should
be used.
References
1. Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for
genome engineering. Cell 157(6):1262–1278
2. Nakagawa M et al (2007) Generation of
induced pluripotent stem cells without Myc
from mouse and human fibroblasts. Nat Biotechnol 26:101
3. Kim D et al (2009) Generation of human
induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell
4(6):472–476
4. Lai T, Yang Y, Ng S (2013) Advances in mammalian cell line development technologies for
recombinant protein production. Pharmaceuticals 6(5):579–603
5. Bu ¨ssow K (2015) Stable mammalian producer
cell lines for structural biology. Curr Opin
Struct Biol 32:81–90
6. Kim TK, Eberwine JH (2010) Mammalian cell
transfection: the present and the future. Anal
Bioanal Chem 397(8):3173–3178
7. Boukany PE et al (2011) Nanochannel electroporation delivers precise amounts of biomolecules into living cells. Nat Nanotechnol 6
(11):747–754
8. Sharei A et al (2013) A vector-free microfluidic
platform for intracellular delivery. Proc Natl
Acad Sci
9. Loh OY et al (2008) Electric field-induced
direct delivery of proteins by a nanofountain
probe. Proc Natl Acad Sci U S A 105
(43):16438–16443
10. Ruiguo Y et al (2018) Monoclonal cell line
generation and CRISPR/Cas9 manipulation
via single-cell electroporation. Small 14
(12):1702495
Nanofountain Probe Electroporation for Monoclonal Cell Line Generation
67
1. The microcontact printing protocol may need to be optimized
if different matrix proteins and substrates are used than those
discussed here. The various substrates, proteins and stamp
polymers used in microcontact printing are discussed in the
following reference [14].
2. In some cases, the contact detection based on the resistance
change might not work due to the presence of air bubbles or
clogging of the probe tip by the transfectant. In such cases a
new pipette with fresh transfectant should be used. The biomolecule concentration can also be lowered to prevent
clogging.
3. If other cells are used, the transfection efficiency may not be
optimal. The pulse parameters and the biomolecule concentration may need to be adjusted to obtain maximum transfection
efficiency. Typically, the voltage amplitude (between 10 V and
25 V) and number of pulses (between 50 and 500) should be
changed to improve transfection.
4. The cell viability may be low in some experiments. This may
be because the pulse profile is too harsh for the cell type. The
voltage amplitude and number of pulses should be reduced in
this case. Keeping the cells for a long time outside the incubator and DNA toxicity due to high plasmid concentrations are
other causes of low viability. Typically, cells should not be kept
outside the incubator for more than 20 min, and lower plasmid concentrations (20 ng/μL to 200 ng/μL) should
be used.
References
1. Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for
genome engineering. Cell 157(6):1262–1278
2. Nakagawa M et al (2007) Generation of
induced pluripotent stem cells without Myc
from mouse and human fibroblasts. Nat Biotechnol 26:101
3. Kim D et al (2009) Generation of human
induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell
4(6):472–476
4. Lai T, Yang Y, Ng S (2013) Advances in mammalian cell line development technologies for
recombinant protein production. Pharmaceuticals 6(5):579–603
5. Bu ¨ssow K (2015) Stable mammalian producer
cell lines for structural biology. Curr Opin
Struct Biol 32:81–90
6. Kim TK, Eberwine JH (2010) Mammalian cell
transfection: the present and the future. Anal
Bioanal Chem 397(8):3173–3178
7. Boukany PE et al (2011) Nanochannel electroporation delivers precise amounts of biomolecules into living cells. Nat Nanotechnol 6
(11):747–754
8. Sharei A et al (2013) A vector-free microfluidic
platform for intracellular delivery. Proc Natl
Acad Sci
9. Loh OY et al (2008) Electric field-induced
direct delivery of proteins by a nanofountain
probe. Proc Natl Acad Sci U S A 105
(43):16438–16443
10. Ruiguo Y et al (2018) Monoclonal cell line
generation and CRISPR/Cas9 manipulation
via single-cell electroporation. Small 14
(12):1702495
Nanofountain Probe Electroporation for Monoclonal Cell Line Generation
67