Facsdiva flow cytometry software version 6

The MMP was analyzed in MCF-7 cells by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Biomol, Hamburg, Germany) staining as previously reported [18 (link)–20 (link)]. Cells (3 mL, 1 × 10⁵ cells/mL) treated for 72 h with different concentrations (¼ × IC₅₀, ½ × IC₅₀ and IC₅₀) of EMW, KCL and doxorubicin (drug control) or DMSO (solvent control) were incubated with JC-1 staining solution for 30 min according to the manufacturer’s protocol, as earlier reported. Subsequently, cells were measured in a BD FACS Aria I Cell Sorter Flow Cytometer (Becton-Dickinson, Germany). The JC-1 signal was measured at an excitation of 561 nm (150 mW) and detected using a 586/15 nm band-pass filter. The signal was analyzed at 640 nm excitation (40 mW) and detected using a 730/45 nm bandpass filter. Cytographs were analyzed using BD FACSDiva™ Flow Cytometry Software Version 6.1.2 (Becton-Dickinson). All experiments were performed at least in triplicates.

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).

The effect of compounds 9 and 13 and doxorubicin (positive control) in cell cycle distribution of MCF-7 cells was performed by flow cytometry using BD cycletest™ Plus DNA Kit Assay (BD Biosciences, San Jose, USA) as previously described [23 (link)]. Cells were tested in 6-well plates (3 mL, 1 × 10⁵ cells/mL) and the incubation time was 72 h in humidified 5% CO₂ atmosphere at 37 °C. The tested concentrations were ¼ × IC₅₀, ½ × IC₅₀ and IC₅₀. Untreated cells (control) were used for comparison with treated cells. The BD FACS Aria I Cell Sorter Flow Cytometer (Becton-Dickinson, Germany) was then used for cell cycle analysis. 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).

The MCF-7 cells were treated with compounds 9 and 13 and doxorubicin, and the integrity of MMP was analyzed using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Biomol, Hamburg, Germany) staining as previously reported [27 (link)–29 (link)]. Cells were tested in 6-well plates (3 mL, 1 × 10⁵ cells/mL) and the incubation time was 72 h in humidified 5% CO₂ atmosphere at 37 °C. The tested concentrations were ¼ × IC₅₀, ½ × IC₅₀ and IC₅₀. Untreated cells (control) were used for comparison with treated cells. JC-1 staining was performed according to the manufacturer’s protocol as reported previously [23 (link)]. Cells were then measured in a BD FACS Aria I Cell Sorter Flow Cytometer (Becton-Dickinson, Germany). The JC-1 signal was measured at an excitation wavelength of 561 nm (150 mW) and detected using a 586/15 nm band-pass filter. The signal was analyzed at an excitation wavelength of 640 nm (40 mW) and detected using a 730/45 nm bandpass filter. Cytographs were analyzed using BD FACSDiva™ Flow Cytometry Software Version 6.1.2 (Becton-Dickinson). All experiments were performed at least in triplicates.