Human brainstem and spinal cord astrocytes

Human brainstem and spinal cord astrocytes (ScienCell) were seeded in 96-well plates until 70–80% confluent and treated with media alone or 10 ng/ml IFNγ with or without ONX 0914 for 48 h. ROS were quantified using the DCFDA Cellular ROS Detection Assay Kit (Abcam), caspase activity was quantified using the Caspase-Glo 3/7 Assay Kit (Promega). Cell viability was detected using the CytoPainter Live Cell Labeling Kit (Abcam) and used to normalize ROS and caspase activity. Plates were read on a Victor 3 Multilabel Counter (Perkin Elmer).

Human brain stem and spinal cord astrocytes (ScienCell) were seeded in six‐well plates until confluent and treated with media alone or recombinant human cytokine for 24 hr. Protein lysate (20 μg) was isolated using RIPA buffer supplemented with a protease and phosphatase‐3 inhibitor cocktail (Sigma‐Aldrich). Lysates were resolved on a 4–12% Tris gel and transferred onto a PVDF transfer membrane (Invitrogen) using an iBlot2 system according to standard protocols. Blots were probed with polyclonal rabbit anti‐VCAM‐1 or ‐CXCR7 (ThermoFisher) and monoclonal mouse anti‐β‐actin (ThermoFisher) antibodies, followed by incubation with appropriate HRP‐conjugated secondary antibodies (ThermoFisher). Blots were imaged using a BioRad ChemiDoc MP imaging system.

Human brainstem and spinal cord astrocytes (ScienCell) were seeded in 6-well plates until 70–80% confluent and treated with media alone or 10 ng/ml IFNγ for 48 h. Protein lysate was isolated in RIPA buffer supplemented with a protease and phosphatase-3 inhibitor cocktail (Sigma-Aldrich). Lysate (20 μg) was then subjected to derivatization according to manufacturer’s instructions (Millipore) and resolved on a 4–12% Tris gel and transferred onto a PVDF transfer membrane (Bio-Rad) using the Trans-Blot Turbo system (Bio-Rad) according to standard protocols. Membranes were incubated overnight at 4 °C in TBST plus 5% powdered milk and probed with either anti-DNP (Millipore; S7150) or anti-β-actin (ThermoFisher Scientific; MA5-15739) primary antibodies, washed with TBST 3 times, and then incubated with HRP-conjugated secondary antibodies (Millipore) for 1 h at room temperature. Membranes were washed with TBST 3 times, imaged using the ChemiDoc MP imaging system (Bio-Rad), and analyzed as previously described [68 (link)].