Pathscan cleaved caspase 3 asp175 sandwich elisa kit

Activation of Caspase-3 by cleavage was performed as a validation for cell death and caspase-3 gene expression. Quantification of cleaved caspase-3 was performed for U87-MG and A172 cells using the PathScan Cleaved Caspase-3 (Asp175) sandwich ELISA kit (Cell Signaling, Danvers, MA). Briefly, after gp120 B/C treatment, the cells were washed with cold PBS and lysed with RIPA buffer (1.5 M Tris pH 8.8, 1.75 g NaCl, 2 mL of 10% sodium 255 dodecyl sulfate, 2 mL Triton X-100; all reagents from Thermo Fisher Scientific, Waltham, MA). Followed by sonication, samples were centrifuged for 10min (x14, 000 rpm) at 4°C. Samples were incubated in the plate for 2 hours at 37°C and washed with 1X wash buffer. Plate was incubated with detection antibody at 37°C for 1 hour, followed by HRP-linked secondary antibody incubation for 30 minutes at 37°C and finalized with TMB substrate incibation for 10 minutes at 37°C. Absorbance was measured and analyzed using SoftMax Pro software (Molecular Devices, Sunnyvale, CA) coupled to a VersaMax Tunable microplate reader (Molecular Devices, Sunnyvale, CA) detecting at 450nm. Positive control for cellular apoptosis was achieved with 100μM Camptothecin diluted in dimethyl sulfoxide (DMSO, Sigma-Aldrich, Saint Louis, MO) for 24 hours.