Cell Cycle Analysis of Anticancer Compounds

Extracts from Passiflora edulis fruit (PEF), Annona muricata leaves (AML), Annona muricata seeds (AMS) that displayed the best cytotoxicity as well as doxorubicin were used to treat CCRF–CEM cells (1 × 10⁶) at their IC₅₀ values. Thus, CCRF–CEM cells were cultured in RPMI medium as described above, in the presence of each sample at a concentration corresponding to the IC₅₀ values obtained in the cell line. The cell cycle was then analyzed after incubation for 24, 48 and 72 h. All reagents, experimental conditions and apparatus were identical to those previously reported (Kuete et al. 2013a (link); Dzoyem et al. 2013 (link)). Briefly, cell cycle analysis was performed by flow cytometry using Vybrant^® DyeCycle™ (Invitrogen, Darmstadt, Germany). Cells were measured after Vybrant^® DyeCycle™ Violet staining (30 min at 37 °C) on a LSR-Fortessa FACS analyzer (Becton–Dickinson, Heidelberg, Germany) using the violet laser. Vybrant^® DyeCycle™ Violet stain was measured with 440 nm excitation. Cytographs were analyzed using FlowJo software (Celeza, Switzerland). All experiments were performed at least in triplicate.