2.1 Solutions
Lock’s solution: 154 mM NsCl, 2.15 mM Na2PO4・2H2O, 10 mM Glucose, 10mM
HEPES, pH = 7.2, 303 mOsm/kg
Sucrose solution: 0.3 M sucrose, 3 mM HEPES, pH = 7.2, 303 mOsm/kg
BCCM (Bovine Chromaffin Complete Medium): Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad CA, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad CA, USA), penicillin/streptomycin (100 IU/ml-100 mg/ml, Invitrogen, Carlsbad CA, USA) and Ara-C (10 μM)(Sigma-Aldrich)
DMEM: Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad CA, USA)
supplemented with 15 mM HEPES, 26 mM NaHCO3, pH = 7.0-7.3, 300 mOsm/kg
PBS: 142 mM NaCl, 2 mM KCl, 8 mM Na2HPO4・2H2O, pH = 7.3, 305 mOsm/kg
Loading buffer: 150 mM NaCl, 5 mM Glucose, 10 mM HEPES, 1mM MgCl2・6H2O, 5 mM KCl, 2.2 mM CaCl2・2H2O, pH = 7.3, 305 mOsm/kg
High K+ solution: 5 mM NaCl, 5 mM Glucose, 10 mM HEPES, 1mM MgCl2・6H2O, 150 mM KCl, 2.2 mM CaCl2・2H2O, pH = 7.3, 305 mOsm/kg
Opti-MEM® I Reduced Serum Medium (Gibco® )
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All other chemicals were reagent grade and were purchased from Sigma-Aldrich, unless indicated specially.
2.2 Primary bovine chromaffin cell culture
I acquired chromaffin cells from bovine adrenal medulla. First, the adipose and
connective tissue surrounding the adrenal gland were removed and the gland was washed by Lock’s solution. The gland was digested by type I collagenase (2 mg/ml dissolved in Lock’s solution; Invitrogen) and incubated for 40 min at 37°C. Then the
gland was opened with surgical scissors and scraped out the medulla with knife. The dissected medulla was filtrated with gauze and the liquid of medulla was centrifuged
(800 G, 5 min). The suspension was discarded and the precipitation was filtrated with 40 μm cell strainer. The precipitation was diluted with Lock’s solution. Again, the
liquid of medulla was centrifuged (800 G, 5 min) and also discard the suspension. The precipitation of medulla cells was collected and mixed with Lock’s solution. To do
density gradient centrifuge, the mixture was loaded on the top of an equal volume sucrose solution carefully. The solution was centrifuged (600G, 5 min). The density gradient centrifuge step was repeated twice. Finally, the cell pellets was resuspended in BCCM, and they were plated at a density of 5 × 105 cell/ml on 24 mm
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collagen-coated coverslips. 3 hours later, when cells adhered to the coverslips better, the dish was added 0.5 ml medium till the total volume was 1.5 ml. I replaced the medium every 2 days.
2.3 Plasmid of NCS-1 and its mutations
The rattus NCS-1 plasmid was obtained from primary cortical neuron in the previous study in our lab. Previous researchers in our lab utilized point mutation to generate NCS-1G2A, NCS-1E120Q and NCS-1R102Q.
Transformation
The 50 ng plasmid was gently mixed with 100 μl competent cell (E. coli, DH5α, ECOSTM 101, Yeastern Biotech, Taiwan) and sat on ice for 30 min. The mixture was
incubated at 42°C for 90 sec and then chilled on ice for 2 min. Next, the mixture was added with 100 μl LB broth and incubated at 37°C and 180 rpm for 45 min. Finally, the mixture was spread on agarose plate containing Ampicillin (100 μg/ml) or Kanamycin (50 μg/ml.) and incubated at 37°C overnight.
Plasmid extraction
Single colonies on agarose plate were picked separately and incubated in 50-ml
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centrifuge tube which had 5 ml LB broth containing 50 μg/ml of Ampicillin or
Kanamycin. The cultures were incubated at 37°C and 180 rpm overnight. To extract
plasmids from the bacteria suspensions, I used QIAprep Spin Miniprep Kit (Qiagen, Germany) according to the manufacture’s manual.
2.4 LTX transfection
I did transfection on day 1-7 after the chromaffin cells were isolated. The protocol was based on the manual of Lipofectamine LTX and Plus Reagent (Invitrogen, Carlsbad CA, USA) with some modifications. First, I mixed 150 μl Opti-MEM®, 2 μl Plus Reagent, and 2 μg plasmid in 1.5-ml eppendorf tube which
wasmarked A;let it stand for 10 min under room temperature. In another 1.5-ml tube, I mixed 150 μl Opti-MEM® with 4μl LTX and marked it as B. The contents of A and
B were mixed and stand for 30 min under room temperature. The medium in the cell culture dish was replaced with Opti-MEM® , and the mixture of plasmid was gently added into the dish. The cells were incubated about 1 day, after that, we replaced the medium with BCCM.
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2.5 Stimulation and Amperometry
I used 150 mM High K+ solution to stimulate single chromaffin cell and measured the released catecholamine by amperometry using ProCFE carbon fiber (Qty-96, Dagan Co., Minneapolis, Minnesota, USA). The puff pipettes were made by Flaming/brown micropipette puller Mode P-97 (Sutter Instrument Co.,Novato, CA, USA) using glass capillary (Catalog 617000, A-M Systems Inc., WA, USA). I utilized PM2000B 4 channel pressure injector (MicroDataInstrument, USA) to puff High K+ solution onto single cells, and utilized HEKA Electronic EPC-10 amplifier (Lambrecht/Pfalz, Germany) and Pulse software (HEKA Electronic, Germany) to measure current. The setting of recording: Gain, 10 mv / pA; Filter 1, Bessel 10 kHz;
Filter 2, Bessel 2.9 kHz.
I put the cells in a recording chamber containing loading buffer. The puff pipette tip was positioned at about 30 μm from the cell and the carbon fiber electrode was
right above the cell with gentle touch (Fig. 1). The High K+ solution in the puff pipette was pressure-puff onto the cells for 3 sec repetitively with an interval of 2 min.
To oxidized the catecholamine, I set the electrode at 650 mV and measured the oxidation current.
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2.6 Calcium imaging technique
To measure the intracellular Ca2+ concentration ([Ca2+]i)_changes, I loaded the cells with Fura-2 AM (0.4 μM, Teflabs, Austin, TX, USA) for 40 min and measured the changes in the fluorescence intensities. I excited the cells with 340 and 380 nm wavelengths alternatively provided by the DG4 (Sutter) and detected the emission by Cool Snap CCD camera (Princeton, US). The whole system was under the control of MetaFlour software (Molecular Devices, Inc., USA). Then it record the emission of the excitation at both 340 nm and 380 nm and created a 340/380 ratio. The High K+ solution in the puff pipette was pressure-puff onto the cells for 3 sec repetitively with an interval of 2 min.
2.7 Data Analysis
The data of amperometry were record by Pulse software and analyzed by MiniAnalysis (Synaptosoft Inc., Chapel Hill, NC, USA). There were the characteristics: amplitude (pA), rise time (ms), decay time (ms), area (fC) and halfwidth (ms) of the elicited spikes to be analyzed. The settings of the analysis:
LoPass filter, 200 Hz; Threshold, 25 pA; Period to search a maximum, 50 ms; Time before a peak for baseline, 75 ms; Period to search a decay time, 200 ms; Fraction of
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peak to find a decay time, 0.37; Period to average a baseline, 20 ms; Area threshold ,60 fC; Number of point to average for peak, 3.
All data were calculated by Excel (Microsoft, USA) and OriginPro (OriginLab, USA). The value of average of the spike characteristic were counted and be compared among groups of cells. Data were presented as mean ± SEM. and analyzed by Kruskal-Wallis ANOVA with Mann-Whitney post hoc test (*: p < 0.05 when compared with the GFP group).
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