Celltiter glo reaction mix

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).

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)).

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.