be adjusted accordingly as recommended by the 4D-Nucleofector™ protocol.
9. It is important to work as quickly as possible. Leaving the cells
in Nucleofector™ Solution for an extended period can lead to
reduced cell viability and transfection efficiency.
10. The amount of the cells and plasmids for transfection can
increase up to fivefold. However, the volume of loaded plasmid
should not exceed 10% of the total volume.
11. To monitor the transient GFP expression in the transfected
cells, we recommend performing flow cytometry analysis. The
expression of GFP should be monitored for at least 2 weeks
after transfection.
12. To obtain a proper growth rate after FACS, the cells should be
seeded at the optimal density as recommended in an appropriate culture vessel.
Acknowledgments
We thank Pa-thai Yenchitsomanus for kindly providing us the K562
and Raji cell lines and Aussara Panya for the NK-92 cell line. This
study was supported by a grant from Siriraj Research Fund, Faculty
of Medicine Siriraj Hospital, Mahidol University (grant number
(IO) R016333015 to MW). M.W. is supported by Chalermphrakiat Grant, Faculty of Medicine Siriraj Hospital, Mahidol University. N.T. is supported by Siriraj Graduate Scholarship, Faculty of
Medicine Siriraj Hospital, Mahidol University, and the Royal
Golden Jubilee Ph.D. Programme.
References
1. Lecoeur H, Fe ´vrier M, Garcia S, Rivie `re Y,
Gougeon M-L (2001) A novel flow cytometric
assay for quantitation and multiparametric
characterization of cell-mediated cytotoxicity.
J Immunol Methods 253(1):177–187.
https://doi.org/10.1016/S0022-1759(01)
00359-3
2. Uzhachenko RV, Shanker A (2019) CD8+ T
lymphocyte and NK cell network: circuitry in
the cytotoxic domain of. Immunity 10:1906.
https://doi.org/10.3389/fimmu.2019.
01906
3. Topham NJ, Hewitt EW (2009) Natural killer
cell cytotoxicity: how do they pull the trigger?
Immunology 128(1):7–15. https://doi.org/
10.1111/j.1365-2567.2009.03123.x
4. Aslantu ¨rk O ¨ S (2018) In vitro cytotoxicity and
cell viability assays: principles, advantages, and
disadvantages. In: ML. Larramendy, & S.
Soloneski (eds). Genotoxicity-A predictable
risk to our actual world, vol 2. InTechOpen,
London, UK. https://doi.org/10.5772/
intechopen.71923
5. Lieberman J (2003) The ABCs of granulemediated cytotoxicity: new weapons in the
arsenal. Nat Rev Immunol 3(5):361–370.
https://doi.org/10.1038/nri1083
6. Zhu Y, Huang B, Shi J (2016) Fas ligand and
lytic granule differentially control cytotoxic
dynamics of natural killer cell against cancer
target.
Oncotarget
7(30):47163–47172.
https://doi.org/10.18632/oncotarget.9980
7. Brunner KT, Mauel J, Cerottini JC, Chapuis B
(1968) Quantitative assay of the lytic action of
immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology 14
(2):181–196
228
Nontaphat Thongsin and Methichit Wattanapanitch
9. It is important to work as quickly as possible. Leaving the cells
in Nucleofector™ Solution for an extended period can lead to
reduced cell viability and transfection efficiency.
10. The amount of the cells and plasmids for transfection can
increase up to fivefold. However, the volume of loaded plasmid
should not exceed 10% of the total volume.
11. To monitor the transient GFP expression in the transfected
cells, we recommend performing flow cytometry analysis. The
expression of GFP should be monitored for at least 2 weeks
after transfection.
12. To obtain a proper growth rate after FACS, the cells should be
seeded at the optimal density as recommended in an appropriate culture vessel.
Acknowledgments
We thank Pa-thai Yenchitsomanus for kindly providing us the K562
and Raji cell lines and Aussara Panya for the NK-92 cell line. This
study was supported by a grant from Siriraj Research Fund, Faculty
of Medicine Siriraj Hospital, Mahidol University (grant number
(IO) R016333015 to MW). M.W. is supported by Chalermphrakiat Grant, Faculty of Medicine Siriraj Hospital, Mahidol University. N.T. is supported by Siriraj Graduate Scholarship, Faculty of
Medicine Siriraj Hospital, Mahidol University, and the Royal
Golden Jubilee Ph.D. Programme.
References
1. Lecoeur H, Fe ´vrier M, Garcia S, Rivie `re Y,
Gougeon M-L (2001) A novel flow cytometric
assay for quantitation and multiparametric
characterization of cell-mediated cytotoxicity.
J Immunol Methods 253(1):177–187.
https://doi.org/10.1016/S0022-1759(01)
00359-3
2. Uzhachenko RV, Shanker A (2019) CD8+ T
lymphocyte and NK cell network: circuitry in
the cytotoxic domain of. Immunity 10:1906.
https://doi.org/10.3389/fimmu.2019.
01906
3. Topham NJ, Hewitt EW (2009) Natural killer
cell cytotoxicity: how do they pull the trigger?
Immunology 128(1):7–15. https://doi.org/
10.1111/j.1365-2567.2009.03123.x
4. Aslantu ¨rk O ¨ S (2018) In vitro cytotoxicity and
cell viability assays: principles, advantages, and
disadvantages. In: ML. Larramendy, & S.
Soloneski (eds). Genotoxicity-A predictable
risk to our actual world, vol 2. InTechOpen,
London, UK. https://doi.org/10.5772/
intechopen.71923
5. Lieberman J (2003) The ABCs of granulemediated cytotoxicity: new weapons in the
arsenal. Nat Rev Immunol 3(5):361–370.
https://doi.org/10.1038/nri1083
6. Zhu Y, Huang B, Shi J (2016) Fas ligand and
lytic granule differentially control cytotoxic
dynamics of natural killer cell against cancer
target.
Oncotarget
7(30):47163–47172.
https://doi.org/10.18632/oncotarget.9980
7. Brunner KT, Mauel J, Cerottini JC, Chapuis B
(1968) Quantitative assay of the lytic action of
immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology 14
(2):181–196
228
Nontaphat Thongsin and Methichit Wattanapanitch