Cell Cycle Analysis of Anticancer Extracts

The cell-cycle analysis was performed by flow cytometry using BD cycletest™ Plus DNA Kit Assay (BD Biosciences, San Jose, USA). The BD Cycletest™ Plus DNA kit provides a set of reagents for isolating and staining cell nuclei. Flow cytometric analysis of differentially stained cells is used to estimate the DNA index (DI) and cell-cycle phase distributions. Briefly, MCF-7 cells (3 mL, 1 × 10⁵ cells/mL) were seeded into each well of 6-well plates and allowed to attach for 24 h. The cells which were treated with ¼ × IC₅₀, ½ × IC₅₀ and IC₅₀ concentrations of Elephantopus mollis whole plant (EMW) and Kalanchoe crenata leaves (KCL) extracts and the standard drug, doxorubicin, and grown for 72 h. The untreated cells (control) were also included in the assay. They were further trypsinized and suspended in 1 mL PBS, then centrifuged at 400 g for 5 min at room temperature (RT). The cells were further processed according to the manufacturer’s protocol [16 (link)]. The cells were further measured on a BD FACS Aria I Cell Sorter Flow Cytometer (Becton-Dickinson, Germany). For each sample 10⁴ cells were counted. For PI excitation, an argon-ion laser emitting at 488 nm was used. Cytographs were analyzed using BD FACSDiva™ Flow Cytometry Software Version 6.1.2 (Becton-Dickinson).