Annexin 5 fitc propidium iodide staining assay

To evaluate the apoptotic rate in GHomas, 150,000 cells/well were plated and cultured for 36 h. Cell cultures were incubated for 12 h with BIM-23A760 as compared with vehicle-treated controls; after that: media were collected, centrifuged 5 min at 1,200 rpm, and the supernatant was discarded and the pellet was maintained; then cells were washed with PBS, detached with a cell scrapper and collected together with the previous pellet. Then, centrifuged for 5 min at 1,200 rpm and the supernatant was discarded while the pellet was processed following manufacturer’s instructions of Annexin-V-FITC/propidium iodide staining assay (Bender Medsystems, Barcelona, Spain) and measurement of apoptotic rate were carried out by flow cytometry (Beckman Coulter, Coulter Epics XL, Madrid, Spain).

To evaluate the apoptotic rate in GH-oma and ACTH-oma cells, 150,000 cells/well were plated and cultured for 36–48 h. Then, cell cultures were incubated for 12 h with AG, acylated In1-19, hydrogen peroxide (used as positive control) and vehicle-treated controls. After the incubation period, culture cells were processed as follows: step-1) media were collected, centrifuged 5 min at 1,200 rpm and, the supernatant was discarded and the pellet was maintained; step-2) cells were washed with PBS, detached using a cell scrapper and collected together with the previous pellet (step-1). Then, the mixture was centrifuged 5 min at 1,200 rpm and the supernatant was discarded while the pellet was processed following manufacturer's instructions of Annexin-V-FITC/propidium iodide staining assay (Bender Medsystems, Barcelona, Spain) and measurement were carried out by flow cytometry (Beckman Coulter, Coulter Epics XL, Madrid, Spain).