Cytofix cytoperm solution

For Annexin‐V/PI staining analysis, DMSO‐ or OTS514‐treated SCLC cells were collected, spun down, washed with PBS, and then resuspended in 50 μL of binding buffer containing 2 μL of APC‐conjugated Annexin‐V antibody (eBioscience, San Diego, CA, USA). After 20 min incubation on ice, the cells were further stained with 100 μL of binding buffer containing 1 μL of propidium iodide (PI) (eBioscience). For cleaved caspase‐3 analysis, SCLC cells were prepared as aforementioned and resuspended in 500 μL of Cytofix/Cytoperm solution (eBioscience). After 20 min incubation on ice, the cells were resuspended with 100 μL of buffer containing 20 μL of PE‐conjugated anti‐cleaved caspase‐3 antibody (eBioscience). For neuronal differentiation detection, prepared SCLC cells were stained with FITC‐conjugated anti‐human CD56 antibody (eBioscience) for 20 min at room temperature. To measure expression levels of CD90 surface protein, cells were stained with APC‐conjugated anti‐human CD90 antibody (5E10) (BD Pharmingen) for 15 min at room temperature. After washing with PBS, samples were subjected to flow cytometry instruments (FACS Calibur or FACS LSRII; Becton Dickinson, San Jose, CA, USA) and analyzed using Flow Jo software (Treestar, Ashland, OR, USA).