26. Add 500 μL of DNase I and incubate for 5 min at 37
C.
27. Let the tissue settle to the bottom of the tube and remove the
trypsin–DNase I mix.
28. Add 8 mL of warm 10%FBS. Gently mix and let tissue settle
again.
29. Remove the 10% FBS and add 10 mL of warm HBSS 1Â. Mix
Gently.
30. Remove the HBSS 1Â and add 3 mL of warm Neuronal
Culture Media-Plating.
31. Dissociate the tissue by pipetting up and down five times using
the fire-polished glass pipettes. Repeat with a second pipette
(see Note 1). Alternatively, the tissue can be dissociated using
the Neuronal Tissue Dissociation Kit—Postnatal Neurons
(Miltenyi Biotec 130-094-802).
32. Collect the cell suspension and filter with a 70 μm cell strainer.
33. Centrifuge the cells at 300 Â g for 10 min and purify the
neurons using the Neuronal Isolation Kit for mouse (Miltenyi
Biotec 130-115-389) following the manufacturer’s instructions. Make sure that neurons are resuspended in Neuronal
Culture Media-Plating.
34. Count the number and concentration of neurons (see Note 2).
Fig. 1 Photographs illustrating the different steps of hippocampal isolation. (a) E18.5 brains are kept on ice-cold
HBSS. (b) A longitudinal cut is made to separate the brains hemispheres. (c) Removal of the noncortical tissue
(arrow) exposes the hippocampus. (d) Hippocampus (arrow) is dissected out from the brain hemisphere
Isolation and Nanoscale Electroporation of Primary Neuronal Cultures In Situ
149
C.
27. Let the tissue settle to the bottom of the tube and remove the
trypsin–DNase I mix.
28. Add 8 mL of warm 10%FBS. Gently mix and let tissue settle
again.
29. Remove the 10% FBS and add 10 mL of warm HBSS 1Â. Mix
Gently.
30. Remove the HBSS 1Â and add 3 mL of warm Neuronal
Culture Media-Plating.
31. Dissociate the tissue by pipetting up and down five times using
the fire-polished glass pipettes. Repeat with a second pipette
(see Note 1). Alternatively, the tissue can be dissociated using
the Neuronal Tissue Dissociation Kit—Postnatal Neurons
(Miltenyi Biotec 130-094-802).
32. Collect the cell suspension and filter with a 70 μm cell strainer.
33. Centrifuge the cells at 300 Â g for 10 min and purify the
neurons using the Neuronal Isolation Kit for mouse (Miltenyi
Biotec 130-115-389) following the manufacturer’s instructions. Make sure that neurons are resuspended in Neuronal
Culture Media-Plating.
34. Count the number and concentration of neurons (see Note 2).
Fig. 1 Photographs illustrating the different steps of hippocampal isolation. (a) E18.5 brains are kept on ice-cold
HBSS. (b) A longitudinal cut is made to separate the brains hemispheres. (c) Removal of the noncortical tissue
(arrow) exposes the hippocampus. (d) Hippocampus (arrow) is dissected out from the brain hemisphere
Isolation and Nanoscale Electroporation of Primary Neuronal Cultures In Situ
149