Pe annexin apoptosis detection kit 1

Tumor cells were suspended in serum-free DMEM/F12 media with 0.5% methylcellulose (Millipore-Sigma), placed in poly-HEMA (Millipore-Sigma) coated plates (1.2 × 10⁵ cells/ml), and incubated for 24 hr. After incubation, the cells were washed twice with PBS, then stained with phycoerythrin-conjugated Annexin V and 7-aminoactinomycin D (7-AAD) using the PE annexin apoptosis detection kit I (BD Pharmingen #559763). A FACSCalibur (BD Bioscience) was used to quantify the apoptotic cells (7AAD^- Annexin V⁺). Single-stained controls were used to gate 7AAD^- and 7AAD⁺ cells while cells suspended for 1 hr were used to define the annexin V⁺ and annexin V^- populations. FlowJo version 9.7.6 (Tree Star) was used to analyze the data (Girnius and Davis, 2017 (link)).