The xCELLigence
RTCA system (Roche) was employed to assess the impact of blocking
A2BAR-mediated effects in cellular impedance upon receptor
activation, as previously described.57 (link)−60 (link) To this end, A2BARSNAP cells were growth in 16-well E-plates (Roche), using DMEM
supplemented with 1 mM sodium pyruvate, 2 mMl -glutamine,
100 U/mL streptomycin, 100 mg/mL penicillin, and 1.5% (v/v) fetal
bovine serum in the presence of 0.5 U/mL of ADA. Of note, wells were
previously coated with 50 μL fibronectin (10 μg/mL; Sigma-Aldrich)
and the background index for each well was determined with supplemented
DMEM (90 μL) in the absence of cells. Subsequently, A2BARSNAP cells (90 μL) were plated at a cell density
of 10,000 cells/well and grown for 18 h in the RTCA SP device station
(Roche) at 37 °C in an atmosphere of 5% CO2. Then,
before ligand addition, cell index values were normalized to the same
time point using the RTCA software, providing the normalized cell
index (NCI). After ligand stimulation, NCI was recorded every 15 s
for a total time of at least 45 min. The area under the curve (AUC)
for every condition was calculated using GraphPad Prism 9.
RTCA system (Roche) was employed to assess the impact of blocking
A2BAR-mediated effects in cellular impedance upon receptor
activation, as previously described.57 (link)−60 (link) To this end, A2BARSNAP cells were growth in 16-well E-plates (Roche), using DMEM
supplemented with 1 mM sodium pyruvate, 2 mM
100 U/mL streptomycin, 100 mg/mL penicillin, and 1.5% (v/v) fetal
bovine serum in the presence of 0.5 U/mL of ADA. Of note, wells were
previously coated with 50 μL fibronectin (10 μg/mL; Sigma-Aldrich)
and the background index for each well was determined with supplemented
DMEM (90 μL) in the absence of cells. Subsequently, A2BARSNAP cells (90 μL) were plated at a cell density
of 10,000 cells/well and grown for 18 h in the RTCA SP device station
(Roche) at 37 °C in an atmosphere of 5% CO2. Then,
before ligand addition, cell index values were normalized to the same
time point using the RTCA software, providing the normalized cell
index (NCI). After ligand stimulation, NCI was recorded every 15 s
for a total time of at least 45 min. The area under the curve (AUC)
for every condition was calculated using GraphPad Prism 9.
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