2.4.1 tsA-201 cell culture
tsA-201 cells (human embryonic kidney 293 cell line, stably expressing SV40 T-antigen) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose and L-glutamine, supplemented with 1 unit/ml penicillin, 1 μg/ml streptomycin, 10 % foetal bovine serum (FBS), and 1 % GlutaMAX (Life
Technologes). The cells were cultured to 80 % confluence in a 5 % CO2
incubator at 37 °C, and passaged every 3 to 4 days.
2.4.2 N2a cell culture
The mouse neuroblastoma cell line Neuro-2a (N2a) was cultured in 50 % DMEM (with high glucose and L-glutamine) and 50 % OPTI-MEM (with L-
glutamine), supplemented with 1 unit/ml penicillin, 1 μg/ml streptomycin, 5 %
FBS, and 1 % GlutaMAX (Life Technologies). The N2a cells were cultured to
2.4.2.1 tsA-201 and N2a cell transfection
To transiently express recombinant proteins, tsA-201 cells were transfected using either PolyJet™ (SignaGen Laboratories) or Fugene® 6 (Promega) in 1:3 ratio with cDNA mix. tsA-201 cells were plated to 30-60% confluent >3 h prior to a transfection procedure. Typically for a 35 mm cell
culture dish, 2 μg cDNA was mixed with 50 μl serum-free DMEM, and 6 μl
PolyJet was mixed with 50 μl serum-free DMEM in separate tubes by pipetting. These were then mixed by gentle pipetting, and incubated for 15 min at room temperature before they were added dropwise to the cells. Similarly for a Fugene-transfection in a 35 mm cell culture dish, 6 μl Fugene was mixed with 94 μl serum-free DMEM, and left to incubate for 5 min at room temperature. 2
μg cDNA mix (typically CaV2.2: β1b: α2δ-1= 3:2:2, in pMT2 vector for tsA-201,
in pcDNA3 or pRK5 for N2a) was then gently mixed with the mixture and incubated for further 30 min before they were added dropwise to the cells.
2.4.3 DRG neuronal cell culture
DRGs were extracted in Hank’s Balanced Salt Solution (HBSS) without
Ca2+ and Mg2+, from the spinal column of a male rat in postnatal Day 10 (P10),
which were killed under the Schedule 1 procedure. DRGs were then dissociated in 1 ml of a dissociation solution (5 mg/ml Dispase (Life Technologies), 2 mg/ml Collagenase (231 U/mg) and 0.1 mg/ml (or 0.1 U/μl) DNase (Life Technologies)
in HBSS without Ca2+ and Mg2+) in a 15 ml falcon tube in a shaking water bath
at 37 °C for 30 min. The enzymes were inactivated by addition of 10% FBS. The cells were triturated by pipetting and centrifuged at 1000 rpm for 3 min to remove the solution. The cells were re-suspended in the culture medium (DMEM/F12, 10 % FBS, 100 ng/ml nerve growth factor (NGF), 1 unit/ml
penicillin, 1 μg/ml streptomycin), which were then plated onto coverslips that
were pre-coated with 17.9 μg/ml poly-L-lysine, and cultured at 37 °C in a 5 %
2.4.3.1 DRG transfection
After the DRGs were dissociated, triturated and centrifuged, the cell pellet was re-suspended in 1 ml HBSS, and split into two Eppendorf tubes (500 μl each) to be further centrifuged at 1000 rpm for 3 min. The cell pellet was re- suspended in 80 μl Nucleofector™ (Lonza) transfection reagent, containing 2
μg of cDNA mix (typically, CaV2.2-pcDNA3: β1b-pRK5: α2δ-1-pcDNA3:
mCherry-pcDNA3= 3:1.5:2:0.5). Following the electroporation protocol in the Nucleofector cuvette, 500 μl of RPMI supplemented with 10 % FBS and 50 ng/ml NGF was added to the cells in the cuvette, which were then transferred
into a 1.5 ml Eppendorf tube and incubated in a 5 % CO2 incubator at 37 °C for
8 min. The cells were plated onto coverslips (250 μl each) that were pre-coated
with poly-L-lysine (Sigma), and cultured at 37 °C in a 5 % CO2 incubator. The
culture medium was changed at 2 h after plating.
2.4.4 DRG-dorsal horn (DH) co-culture
DRGs and DH were extracted in ice-cold L15 dissection medium, from a male rat at postnatal day 0 (P0), which was killed under the Schedule 1 procedure. The DH was dissociated in 100 μl 2.5% trypsin in S-MEM at 37 °C for 20-25 min in a dish. The dissociated DH was transferred into a 15 ml tube and was washed twice with warm culture medium (50 % NeuroBasal A (Life Technologies), 2 % B27-supplement (Life Technologies), 100 ng/ml NGF, 1
unit/ml penicillin, 1 μg/ml streptomycin, 1 % Glutamax, 50 % astrocytes-
conditioned medium*), and triturated gently using a fire polished glass pipette. DH neurons were plated onto coverslips coated with poly-L-lysine and laminin (Sigma). DRGs were dissociated in 1 ml of 5 mg/ml Dispase at 37 °C for 25-30 min in a dish. The dissociated DRGs were transferred to a 1.5ml Eppendorf tube and triturated with pipettes. The supernatant was removed by centrifuging at 1000 rpm for 3 min. The pellet was washed with 1 ml of warm HBSS and centrifuged again. For DRG transfection, the supernatant was discarded and the cells were resuspended in 80 μl Nucleofector™ (Lonza) transfection reagent, and they were transferred to a fresh tube containing 2 μg of cDNA mix
(HA-CaV2.2-pcDNA3: β1b-pRK5: α2δ-1-pcDNA3: vGpH-pcDNA3= 3:1:1.5:1.5).
RPMI supplemented with 10 % FBS and 50 ng/ml NGF was added to the cells in the cuvette, which were then transferred into a 1.5 ml Eppendorf tube and
incubated in a 5 % CO2 incubator at 37 °C for 8 min. The cells were plated on
top of the DH neurons. The wells were flooded with warm medium 1 h after the transfected DRG neurons were plated. The growth medium was replaced after 16h, and half of the medium every 3-4 days. Cells were cultured for 7 days before the experiment.
(*Astrocytes-conditioned medium was prepared from growth medium of the astrocytes culture from dissociated cerebral cortex of P0 rat. The growth medium contained DMEM with 10 % horse serum, 2.5 % FBS, 1 unit/ml penicillin, 1 μg/ml streptomycin.)
2.4.5 Hippocampal neuronal cell culture
Hippocampal neurons were isolated from the hippocampus of P0 rats, which were killed under the Schedule 1 procedure. The cerebrum was cut into two, and the hippocampi were extracted from each hemisphere in ice cold HBSS with 10 mM HEPES. The hippocampi were then cut into small segments and digested gently in Papain solution (7 U/ml Papain, 0.2 mg/ml L-Cysteine, 0.2 mg/ml Bovine Serum Albumin (BSA), 5 mg/ml glucose, 10 mM HEPES in
HBSS with 200 μl DNase) in a shaking water both for 40 min at 37 °C. The
cells were washed twice with the growth medium (NeuroBasal, 2 % B27-
supplement, 1 unit/ml penicillin, 1 μg/ml streptomycin, 1 % Glutamax, 0.1% β-
marcaptoethanol), and triturated gently. The cells were plated onto the coverslips that are coated with poly-L-lysine and laminin at 750 cell/μl, 100 μl per coverslip. The entire growth medium was changed after 2 h of plating, and half of the medium was then changed every 3-4 days.