2.3 NanochannelBased Electroporation
(NEP)
NEP platforms consist of an assembly of modified 12-mm diameter
polyester transwell inserts with 0.4 μm pore size (Corning Inc.) and
a polydimethylsiloxane (PDMS) reservoir gasket placed on top gold
sleeve, together with a gold-plated electrode with plastic casing
(Thermo Scientific Inc.) connected to the electrodes of a Gene
Pulser Xcell power source (Bio-Rad Laboratories) [11].
3 Methods
3.1 Reagent
Preparation
1. Boric acid 100 mM, pH 8.2: Dilute 309.15 mg of boric acid
(MW 61.83 g/mol) in 40 mL of ultrapure water, set pH to 8.2
and complete volume to 50 mL.
2. Boric acid 166.3 mM, pH 8.2: Dilute 513.189 mg in 40 mL of
ultrapure water, set pH to 8.2 and complete volume to 50 mL.
3. 10Â stock poly-D-lysine (PDL) (1 mg/mL): Dilute 50 mg of
poly-D-lysine in boric acid (100 mM, pH 8.2). Make 1.5 mL
aliquots, keep at À20
C.
4. 1Â PDL (0.1 mg/mL): Dilute the 1.5 mL of 10Â stock to a
volume of 15 mL 1Â (0.1 mg/mL) with Boric Acid 166.3 mM
pH 8.2, and filter it with Cellulose Acetate Syringe Filters
0.2 μm to sterilize. Store at À20
C
5. DNase I: Dilute 100 mg in 10 mL of HBSS 1Â and make
500 μL aliquots. Store at À20
C.
6. 10% FBS: Dilute the heat-inactivated FBS (1:10) in HBSS.
Make 10 mL aliquots and store at 4
C.
7. Glutamate 10 mM: Dilute 93.565 mg of glutamate
(MW187.130 g/mol) in 50 mL of ultrapure water. Filter it
with cellulose acetate syringe filters 0.2 μm to sterilize.
8. Neurocult SM1-supplement: Make aliquots of 1 mL and store at
À20
C.
9. Neuronal Culture Media-Plating: Mix 500 mL of GlutaMax
(100Â), 1 mL of SM1-supplement and 125 mL of glutamate
10 mM and complete the volume to 50 mL with Neurobasal™
medium.
3.2 Neuronal Culture
Preparation
Two days before culture day:
1. Set and autoclave surgery tools.
2. Fire-polish the tips of glass Pasteur pipettes to get a smooth
opening of about 1.5–2 mm and autoclave.
3. Cover the polyester transwell inserts with 1Â PDL. Incubate
overnight in a humidified incubator.
One day before culture day
4. Remove PDL solution from the culture inserts and wash with
sterile water for 30 min, repeat this washing step three times.
Isolation and Nanoscale Electroporation of Primary Neuronal Cultures In Situ
147
(NEP)
NEP platforms consist of an assembly of modified 12-mm diameter
polyester transwell inserts with 0.4 μm pore size (Corning Inc.) and
a polydimethylsiloxane (PDMS) reservoir gasket placed on top gold
sleeve, together with a gold-plated electrode with plastic casing
(Thermo Scientific Inc.) connected to the electrodes of a Gene
Pulser Xcell power source (Bio-Rad Laboratories) [11].
3 Methods
3.1 Reagent
Preparation
1. Boric acid 100 mM, pH 8.2: Dilute 309.15 mg of boric acid
(MW 61.83 g/mol) in 40 mL of ultrapure water, set pH to 8.2
and complete volume to 50 mL.
2. Boric acid 166.3 mM, pH 8.2: Dilute 513.189 mg in 40 mL of
ultrapure water, set pH to 8.2 and complete volume to 50 mL.
3. 10Â stock poly-D-lysine (PDL) (1 mg/mL): Dilute 50 mg of
poly-D-lysine in boric acid (100 mM, pH 8.2). Make 1.5 mL
aliquots, keep at À20
C.
4. 1Â PDL (0.1 mg/mL): Dilute the 1.5 mL of 10Â stock to a
volume of 15 mL 1Â (0.1 mg/mL) with Boric Acid 166.3 mM
pH 8.2, and filter it with Cellulose Acetate Syringe Filters
0.2 μm to sterilize. Store at À20
C
5. DNase I: Dilute 100 mg in 10 mL of HBSS 1Â and make
500 μL aliquots. Store at À20
C.
6. 10% FBS: Dilute the heat-inactivated FBS (1:10) in HBSS.
Make 10 mL aliquots and store at 4
C.
7. Glutamate 10 mM: Dilute 93.565 mg of glutamate
(MW187.130 g/mol) in 50 mL of ultrapure water. Filter it
with cellulose acetate syringe filters 0.2 μm to sterilize.
8. Neurocult SM1-supplement: Make aliquots of 1 mL and store at
À20
C.
9. Neuronal Culture Media-Plating: Mix 500 mL of GlutaMax
(100Â), 1 mL of SM1-supplement and 125 mL of glutamate
10 mM and complete the volume to 50 mL with Neurobasal™
medium.
3.2 Neuronal Culture
Preparation
Two days before culture day:
1. Set and autoclave surgery tools.
2. Fire-polish the tips of glass Pasteur pipettes to get a smooth
opening of about 1.5–2 mm and autoclave.
3. Cover the polyester transwell inserts with 1Â PDL. Incubate
overnight in a humidified incubator.
One day before culture day
4. Remove PDL solution from the culture inserts and wash with
sterile water for 30 min, repeat this washing step three times.
Isolation and Nanoscale Electroporation of Primary Neuronal Cultures In Situ
147