Lsr 2 flow cytometry analyzer

Since the pure compounds tested were more effective on HCT 116 cells, apoptosis induction was determined for these cells. For apoptosis measurement, HCT 116 cells were seeded at 3 × 10⁵ cells per well in a six-well plate as described before^{17 (link)}. After 24 h, they were treated with D3G, DC, GA, red grape combination (RGC: D3G, M3G, and Q3G (57:33:10), and oxaliplatin for 24 h at the IC₅₀ determined values (ranging from 13.5–395.8 µg/mL). A BD LSR II Flow Cytometry Analyzer (BD Biosciences, San Jose, CA) was used to evaluate the state of 10,000 cells. Duplicate readings of each well were taken, and two independent experiments were analyzed. Total apoptotic cells was a combination of late and early apoptotic cells in quadrants two and four, respectively analyzed through FCS Express 5 Software.

Mitophagy was assessed using MitoTracker Red CMXRos (MTR; Molecular Probes) and TO (BD Biosciences) as described (Zhang et al., 2009 (link)). Doubly stained reticulocytes were obtained by sequential staining with MTR, then TO. MTR fluorescence emission between 600 and 620 nm was collected after 562 nm laser excitation using a BD LSR II flow cytometry analyzer (BD Biosciences).

DCF fluorescence helps to measures innumerable ROS such as H₂O₂ and hydroxyl radicals^{49 (link)}. That why DCFDA staining was used to quantify the reactive oxygen species (ROS) level inside the cell as described earlier^{17 (link),41 (link)}. Briefly, the MCF-7 cells (70–80% confluent) were incubated with IC₅₀ concentration of each compound and positive control H₂O_2, respectively in a 24-well culture plates. After 5–6 h incubation of cells with respective compounds, cells were gently washed with 500 μl of prewarmed (at 37 °C) Krebs Ringer buffer (20 mM HEPES, 2 mM MgSO₄, 10 mM dextrose, 127 mM NaCl, 1 mM CaCl₂ and 5.5 mM KCl), subsequently 10 µM DCFDA (Invitrogen Grand Island, NY) has been added to each well and incubated further for 30 min in dark at 37 °C in a humidified CO₂ incubator. After 30 min incubation, the cells were collected by trypsinization and ROS levels were quantified with the help of Flow Cytometry. Nearly 10,000 events for each sample were collected on BD LSR II Flow Cytometry Analyzer, and analyzed with help of FlowJo. For intact cell imaging, cells were washed with PBS, pH 7.4 and imaged for ROS levels estimation using the DCFDA fluorescent dye. Fluorescence images were taken on Nikon-EclipseTS100 microscope.

Annexin-V staining was used to assess the apoptotic potential of the selected hydrazones as described.^{22,41 (link)} Briefly, cells were treated with IC₅₀ concentration of selected hydrazones for 24 h and control cells were treated with DMSO/culture medium only. After 24 h incubation, approximately 2.0–2.5 × 10⁶ cells were collected by trypsinization and washed twice with PBS (5–10 mL). Finally, FITC labelled Annexin-V and PE labelled PI was used for staining the cells using the FITC-Annexin-V kit by following manufacture instructions (BD-Biosciences, USA). Nearly, 10 000 events were recorded using BD LSR II Flow Cytometry Analyzer and analyzed by FlowJo software.