DNase I. If the issue persists, check the opening of the glass
pipette; if the opening is too big, it will be hard to get a single
cell suspension.
2. Viability during cell counting should be !80%. If the viability
drops below 80% the most likely cause is a harsh dissociation.
To avoid this, try to reduce the number of times that you pipet
up and down with the glass Pasteur pipettes. This issue could
also occur if the opening on the glass pipette is too narrow. To
correct this, leave a bigger opening when fire-polishing the
pipettes. If the viability issues persist, check the pH of the
HBSS 1Â.
3. If neurons do not adhere to the well/coverslips, check the
concentration and time of incubation of PDL 1Â as well as
the pH and concentration of the boric acid. Also, the coating
procedure should not be performed more than 1 week in
advance. If the problem persists, check the concentration of
glutamate.
4. If cultures get contaminated, make sure that surgical tools are
properly autoclaved and clean all the working surfaces with 70%
ethanol.
5. If the viability of cells decreased after 3–4 DIV, check the media
composition, making sure that all components are fresh. Neurons release essential growth factors, so remember to change
only one-third of the media volume once a week. Minimize the
time that neuronal cultures are outside the incubator by changing the media of one plate at a time.
References
1. Schlachetzki F, Zhang Y, Boado RJ, Pardridge
WM (2004) Gene therapy of the brain: the
trans-vascular
approach.
Neurology
62:1275–1281.
https://doi.org/10.1212/
01.wnl.0000120551.38463.d9
2. Kaech S, Banker G (2006) Culturing hippocampal neurons. Nat Protoc 1:2406–2415.
https://doi.org/10.1038/nprot.2006.356
3. Lundstrom K (2018) Viral vectors in gene
therapy. Diseases 6. https://doi.org/10.
3390/diseases6020042
4. Mehier-Humbert S, Guy RH (2005) Physical
methods for gene transfer: improving the
kinetics of gene delivery into cells. Adv Drug
Deliv Rev 57:733–753. https://doi.org/10.
1016/j.addr.2004.12.007
5. Fei Z, Wu Y, Sharma S, Gallego-Perez D,
Higuita-Castro N, Hansford D et al (2013)
Gene delivery to cultured embryonic stem
cells
using
nanofiber-based
sandwich
electroporation. Anal Chem 85:1401–1407.
https://doi.org/10.1021/ac302140p
6. Chang L, Gallego-Perez D, Zhao X, Bertani P,
Yang Z, Chiang C-L et al (2015)
Dielectrophoresis-assisted 3D nanoelectroporation for non-viral cell transfection in adoptive immunotherapy. Lab Chip 15:3147–3153.
https://doi.org/10.1039/c5lc00553a
7. Zhao X, Wu Y, Gallego-Perez D, Kwak KJ,
Gupta C, Ouyang X et al (2015) Effect of
nonendocytic uptake of nanoparticles on
human bronchial epithelial cells. Anal Chem
87:3208–3215.. 10.1002/advs.201500111.
https://doi.org/10.1021/ac503366w
8. Zhao X, Huang X, Wang X, Wu Y, Eisfeld A-K,
Schwind S et al (2015) Nanochannel electroporation as a platform for living cell interrogation in acute myeloid leukemia. Adv Sci
2:1500111. https://doi.org/10.1002/advs.
201500111
Isolation and Nanoscale Electroporation of Primary Neuronal Cultures In Situ
151
pipette; if the opening is too big, it will be hard to get a single
cell suspension.
2. Viability during cell counting should be !80%. If the viability
drops below 80% the most likely cause is a harsh dissociation.
To avoid this, try to reduce the number of times that you pipet
up and down with the glass Pasteur pipettes. This issue could
also occur if the opening on the glass pipette is too narrow. To
correct this, leave a bigger opening when fire-polishing the
pipettes. If the viability issues persist, check the pH of the
HBSS 1Â.
3. If neurons do not adhere to the well/coverslips, check the
concentration and time of incubation of PDL 1Â as well as
the pH and concentration of the boric acid. Also, the coating
procedure should not be performed more than 1 week in
advance. If the problem persists, check the concentration of
glutamate.
4. If cultures get contaminated, make sure that surgical tools are
properly autoclaved and clean all the working surfaces with 70%
ethanol.
5. If the viability of cells decreased after 3–4 DIV, check the media
composition, making sure that all components are fresh. Neurons release essential growth factors, so remember to change
only one-third of the media volume once a week. Minimize the
time that neuronal cultures are outside the incubator by changing the media of one plate at a time.
References
1. Schlachetzki F, Zhang Y, Boado RJ, Pardridge
WM (2004) Gene therapy of the brain: the
trans-vascular
approach.
Neurology
62:1275–1281.
https://doi.org/10.1212/
01.wnl.0000120551.38463.d9
2. Kaech S, Banker G (2006) Culturing hippocampal neurons. Nat Protoc 1:2406–2415.
https://doi.org/10.1038/nprot.2006.356
3. Lundstrom K (2018) Viral vectors in gene
therapy. Diseases 6. https://doi.org/10.
3390/diseases6020042
4. Mehier-Humbert S, Guy RH (2005) Physical
methods for gene transfer: improving the
kinetics of gene delivery into cells. Adv Drug
Deliv Rev 57:733–753. https://doi.org/10.
1016/j.addr.2004.12.007
5. Fei Z, Wu Y, Sharma S, Gallego-Perez D,
Higuita-Castro N, Hansford D et al (2013)
Gene delivery to cultured embryonic stem
cells
using
nanofiber-based
sandwich
electroporation. Anal Chem 85:1401–1407.
https://doi.org/10.1021/ac302140p
6. Chang L, Gallego-Perez D, Zhao X, Bertani P,
Yang Z, Chiang C-L et al (2015)
Dielectrophoresis-assisted 3D nanoelectroporation for non-viral cell transfection in adoptive immunotherapy. Lab Chip 15:3147–3153.
https://doi.org/10.1039/c5lc00553a
7. Zhao X, Wu Y, Gallego-Perez D, Kwak KJ,
Gupta C, Ouyang X et al (2015) Effect of
nonendocytic uptake of nanoparticles on
human bronchial epithelial cells. Anal Chem
87:3208–3215.. 10.1002/advs.201500111.
https://doi.org/10.1021/ac503366w
8. Zhao X, Huang X, Wang X, Wu Y, Eisfeld A-K,
Schwind S et al (2015) Nanochannel electroporation as a platform for living cell interrogation in acute myeloid leukemia. Adv Sci
2:1500111. https://doi.org/10.1002/advs.
201500111
Isolation and Nanoscale Electroporation of Primary Neuronal Cultures In Situ
151