After passaging, PC12 cells were seeded into 96-well plates (5×103/well; 200 µl/well) and incubated in an environment with 5% CO2 at 37°C for 24 h. These cells were divided into two groups: L-DOPA treatment group and CD39 inhibitor pre-treatment group (cells were pre-incubated with 0.5 mM ARL 67156 [6-N, N’-diethyl-D-β-γ-dibromomethylene ATP, Santa Cruz USA] for 30 min or 10 µM H89 [a PKA inhibitor] for 1 h before L-DOPA treatment). The nucleotide analogue ARL 67156 is a selective inhibitor of ecto-ATPase of CD39/NTPDase family [69]. Cells were then treated with L-DOPA at different concentrations (0, 1, 5, 10 and 20 µmol/L) for 3 days. On the fourth day, cells were incubated with 100 µmol/L H2O2 for 8 h. Finally, MTT assay and LDH assay were performed to determine the viability of PC12 cells. The oxidative stress induced by H2O2 was evaluated by detection of ROS.
Arl 67156
Manufactured by Santa Cruz Biotechnology
Sourced in United States
ARL-67156 is a laboratory product manufactured by Santa Cruz Biotechnology. It is a chemical compound used in research applications. No further details about its core function or intended use can be provided in an unbiased and factual manner.
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Lab products found in correlation
4 protocols using arl 67156
1
Cytoprotective Effects of L-DOPA on PC12 Cells
2
Astrocyte ATP Release Quantification
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Twelve thousand primary astrocytes were seeded in 48-well plates. After 24 h, cells were incubated in serum-free media containing 100 μM of the exonuclease inhibitor ARL-67156 (Santa Cruz Biotechnologies) for 30 min at 37 °C. Following this, cells were stimulated with Thy-1-Fc:Protein A for 15 min. Where indicated, cells were incubated with a combination of Heptanol (Sigma-Aldrich Co.) (500 μM) and Probenecid (Sigma-Aldrich Co.) (1 mM). Next, culture medium was recovered and centrifuged for 5 min at 1000×g. The supernatants were incubated in the dark with 50 μl CellTiter-Glo® reaction mix (Promega, Madison, WI). Luminescence intensity was determined in a Synergy2 multi-mode reader (Biotek Instruments, Inc., Vermont), and the values were interpolated using a calibration curve obtained from different ATP concentrations (0.01, 0.1, 1, and 10 μM).
3
ATP-based Viability Assay in MDA-MB-231 Cells
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MDA-MB-231 cells (1 × 104) were seeded in 48-well plates. After 24 h, cells were incubated in serum-free media containing 100 μM of the exonuclease inhibitor ARL-67156 (Santa Cruz Biotechnology, CA, United States) for 30 min, at 37°C. Cells were then stimulated with 8 μg of Thy-1-Fc coupled to Protein A (0.8 μg) for different time periods. Next, 50 μl of culture medium were recovered and incubated in the dark with 50 μl of CellTiter-Glo® reaction mix (Promega, Madison, WI, United States). Luminescence intensity was determined in a Synergy2 multi-mode reader (Biotek Instruments, Inc., Winooski, VT, United States) and the values were interpolated using a calibration curve obtained with different ATP concentrations (0.01, 0.1, 1, and 10 μM) (Henriquez et al., 2011 (link)).
4
Astrocyte Extracellular ATP Quantification
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Primary astrocytes (5 × 104) were seeded per well in 48-well plates. After 24 h, cells were incubated in SFM containing 100 μM of the exonuclease inhibitor ARL-67156 (Santa Cruz Biotechnologies, Dallas, TX, USA) for 30 min at 37 °C. Then, the cells were stimulated with 2 µg of Thy-1-Fc per well, for 10 min. Where indicated, cells were incubated for 30 min with an AKT-inhibitor (3 µM AKTi, AKT inhibitor VIII, Merck Millipore, Burlington, MA, USA) or a PI3K-inhibitor (3 µM LY294002, Sigma-Aldrich Co., St. Louis, MO, USA). Next, the culture medium was recovered and centrifuged for 5 min at 1000 × g. The supernatants were incubated in the dark with 50 μl CellTiter-Glo® reaction mix (Promega, Madison, WI, USA). Luminescence intensity was determined in a Synergy2 multi-mode reader (Biotek Instruments, Inc., Winooski, VT, USA), and the values were calculated using a calibration curve obtained with ATP concentrations of 0.01, 0.1, 1 and 10 μM.
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