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Functional characterization of P2Y1 and P2X4 receptors in human neuroblastoma SK-N-MC cells
Collection (ATCC, Barcelona, Spain). Cells were grown in cases, antibodies against the rat protein were also used (if
monolayer in 10-cm plastic culture dishes (BD Falcon, it was the case, cross reactivity with the human protein has
Erembodegem, Belgium) at 37 ºC in a humidified been verified by the manufacturer). Next, covers were
atmosphere of 95% air and 5% CO2. Culture medium was washed three times with PBS/BSA and incubated for 1 h at
minimum essential medium (Gibco, Rockville, MD, USA) 37ºC with the secondary antibodies: donkey anti-rabbit
supplemented with 10% heat-inactivated fetal calf serum IgG coupled to Cy3 (1:400) (Jackson Immunoresearch,
(Gibco), 2 mM glutamine, 1 mM sodium pyruvate, non- West Grove, PA, USA) or fluorescein conjugated goat anti
essential amino acids (alanine, glutamic acid, aspartic acid, guinea-pig IgG (1:64) (Sigma). Then, covers were washed
proline and asparagine, each at 0.4 mM), 100 units/mL three times with PBS and mounted following standard
penicillin, 0.1 mg/ml streptomycin and 0.25 µg/mL procedures. Control experiments of nonspecific staining
amphotericin B (Sigma). were performed without primary antibody or by
preadsorption of the primary antibody with the
Cells were subcultured about 2 times a week: culture immunizing peptide.
medium was removed and discarded and the cell layer was
rinsed two times with 2 mL of a solution containing 0.25% Fluorescent images were taken using a NIKON TE-200
(w/v) trypsin and 0,02% (w/v) EDTA in Hank's Balanced microscope equipped with a S Fluor 40X objective (oil,
Salt Solution with phenol red (Sigma). Then, culture 1.3 NA) a mercury lamp light source, fluorescein and
dishes were placed in an incubator for 1 min at 37º to rhodamine Nikon filter sets and a CCD camera (Kappa
allow cells to detach. After that, 5 mL of the culture ACC 1) controlled by Kappa ImageBase software (Kappa
medium were added to the dishes and cells were aspirated opto-electronics, Gleichen, Germany).
by gently pippeting. Cells were counted in a Neubauer
chamber using the trypan blue exclusion test and 2.4. W estern blot
appropriate aliquots of the cell suspension were taken for
the immunocitochemical, western blot or calcium imaging Cells were seeded on 10-cm plastic Petri dishes (BD
assays. Finally, 1 mL of the cell suspension was added to a Falcon) at a density of 400,000 cells/mL and maintained at
new culture dish containing 9 mL of the culture medium 37 ºC in 5% CO2 for 2-3 days before any treatment.
and cells were maintained at 37°C in 5% CO2. Western blot analysis were performed using protein
extracted from the SK-N-MC cells by homogenization in a
2.3. Immunocytochemical assays buffer containing 100 nM HEPES (pH 7.4), 0.5 mM
EDTA, 50 µg/ml phenylmethylsulfonyl fluoride, 0.5 µg/ml
Cells were plated at a density of 200,000 cells/mL on leupeptine, 0.5 µg/ml pepstatine, 0.2 M NaCl and 1% (v/v)
glass coverslips precoated with 0.1 mg/mL poly-L-lysine Triton X-100. The samples were homogenized at 4°C and
(Biochrom AG, Berlin Germany) and maintained in a protein content determined by Bradford assay. Total
humidified incubator at 37 ºC in 5% CO2 for 2-3 days protein (40 µg) was electrophoresed on 10% SDS-
before they were used in the immunocytochemical polyacrylamide gels and transferred to nitrocellulose
experiments. Cover slips with the SK-N-MC cells attached membranes (Whatman, Maidestone, UK). The membranes
were then treated with 4% (w/v) p-formaldehyde (Sigma) were blocked with 5% nonfat dried milk for 1 h at room
for 15 min, washed twice with phosphate-buffered saline temperature and further incubated overnight at 4ºC with
(PBS) (composition mM: NaCl 137, KCl 2.6, KH2PO4 1.5, the different P2 receptor antibodies at a dilution of 1:500.
Na2HPO4 8.1, pH 7.4) and incubated for 1 h in PBS Blots were then washed and incubated for 1 h at room
containing 3% (w/v) bovine serum albumine (BSA), 0.1% temperature with the corresponding secondary antibody
(v/v) Triton X-100 and 5% (v/v) normal goat or donkey coupled to horseradish peroxidase (DakoCytomation,
serum (Sigma). Cells were washed three times with Glostrup, Denmark). Protein bands were detected with
PBS/BSA and incubated with the primary antibody for 1 h enhanced chemoluminiscence detection (Perkin Elmer,
at 37º C. Primary antibodies assayed were: rabbit anti- Boston, MA, USA). Incubation with the control peptide
P2Y1 (serum at 1:100 dilution) (Alomone labs, Jerusalem, was made following the manufacturer instructions.
Israel), rabbit anti-P2Y2 (1:200) (Chemicon International,
Temecula, CA, USA), rabbit anti-P2Y4 (1:100) (Chemicon 2.5. Calcium imaging
International), rabbit anti-P2Y6 (1:100) (Alomone labs),
rabbit anti-P2Y11 (1:100) (Alomone labs), rabbit anti- Cells were plated at a density of 200,000 cells/mL on
P2Y12 (1:100) (Alomone labs), rabbit anti P2Y13 (1:50) glass coverslips precoated with 0.1 mg/mL poly-L-lysine
(Alomone labs), rabbit anti-P2X1 (1:100) (Chemicon (Biochrom) and maintained in a humidified incubator at 37
International), guinea-pig anti-P2X2 (1:250) (Chemicon ºC in 5% CO2 for 2-3 days before they were used in the
International), guinea-pig anti-P2X3 (1:500) (Chemicon calcium imaging experiments. Cells attached to coverslips
International), rabbit anti-P2X4 (1:100) (Alomone labs), were then washed with Locke’s solution (composition in
rabbit anti-P2X5 (1:200) (Alomone labs), rabbit anti-P2X6 mM: NaCl, 140; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.2;
(1:100) (Alomone labs) or rabbit anti-P2X7 (intracellular CaCl2, 2.5; glucose, 5.5; HEPES, 10; pH 7.4)
epitope, 1:100 dilution) (Alomone labs). All the antibodies supplemented with 1 mg/mL BSA and loaded with 5 µM
used in this work were polyclonal and most of them were Fura-2 AM (Molecular Probes, Eugene, OR, USA) for 30
raised against the corresponding human protein. In some min at 37 ºC. Next, the coverslips were washed with
Locke’s medium and mounted in a small superfusion
@Real Academia Nacional de Farmacia. Spain 249
