br Fluorescence microscopy examination for nuclear localizat
2.13. Fluorescence microscopy examination for nuclear localizations
Examination of the nuclear fragmentation and nuclear localizations of both untreated and treated cells with the selective compounds, mixtures and their complex were carried out by DAPI staining (Table 1). The HeLa cells were grown on 6-well plates (1 × 105 cells/ well) and sustained at 37 °C with 5% CO2 for 48 h under humidified condition. Then, the cancer cells were treated with sub-IC50 con-centrations of compounds and Ru-Fu complex for 24 h. After
IC50 values of rutin (Ru), fucoidan (Fu) and Ru-Fu complex against HaCaT and HeLa cancer cells. Selection of sub-IC50 and IC50 concentration of complex in this study.
Compounds HaCaT HeLa Selected sub-IC50 Concentrations Concentrations used for this study
incubation, cells were washed with PBS and fixed in Conroy's fixative for 20 min. Further, the cells were re-washed using methanol and stained with DAPI (10 μg/ml) at room temperature for 20 min. in dark. The fragmented apoptotic nuclei were viewed using fluorescent mi-croscopy in appropriate wavelengths (340–380 nm).
2.14. Assay of cell apoptosis and quantification
Flow cytometry was used to assess quantitatively the extent of apoptosis upon treatment of HeLa cells with the study samples. In brief, the cells were pre-treated with rutin alone, fucoidan alone, Ru + Fu mixture and Ru-Fu complex (Table 1). The treated cells were suspended in 200 μl of binding buﬀer, and the suspension was added with 10 μl of annexin-V-FITC and 5 μl of PI followed by incubation for 15 min in the dark at room temperature. Subsequently, 300 μl of binding buﬀer was added to the cell suspension and the cells were analyzed by flow cyt-ometer (BD, FACS Calibur, USA).
2.15. Flow cytometric analysis for Tunicamycin distribution
The potential of rutin, fucoidan, Ru + Fu mixture and Ru-Fu com-plex to arrest cell cycle (Table 1) in HeLa cells was assessed by flow cytometry. The HeLa cells (1 × 106) were seeded in each tissue culture dish and allowed to attach overnight. The cells were treated with rutin, fucoidan, Ru + Fu mixture and Ru-Fu complex for 24 h. The cells were harvested using trypsinization followed by pelleting at 2500 rpm for 5 min at room temperature. Then, the cells were re-suspended in 300 μl of PBS-EDTA to which 700 μl of chilled 70% ethanol were added drop-wise with slow mixing. The solution was added gently to ensure com-plete mixing of ethanol, and the samples were stored at °C overnight. Then, 1:100 volumes of (20 μg/ml) RNase were added, and the mixture was incubated at 37 °C for 1 h. Propidium iodide (PI) was added (50 μg/ ml) and incubated for 10–20 min at RT. The stained cells were analysed for DNA histograms and cell cycle phase distribution using flow cyto-metry (Becton Dickinson Immuno-cytometry System; BD, Franklin Lakes, NJ).
2.16. In vitro hemolysis assay
In vitro hemolysis assay was performed as described by Rajiu et al. . Briefly, fresh blood samples were sourced from healthy volunteers and stabilized with ethylene-diamine tetra acetic acid (EDTA). 2 ml of blood sample was added to 4 ml of sterile isotonic phosphate-buﬀered saline (PBS) and centrifuged at 5000 rpm for 10 min to separate RBCs from serum. The RBCs were purified by washing, re-suspended with PBS for five times, and then diluted to 20 ml using PBS. To test he-molytic activity, 0.2 ml of diluted RBC suspension was added to 0.8 ml of Ru-Fu complex in PBS at diﬀerent concentrations (2.5, 5, 10, 25, 50 and 100 μg). Positive and negative controls were obtained by sus-pending RBCs in deionized water and PBS, respectively. The samples were incubated at room temperature for 3 h, followed by centrifugation at 10,000 rpm for 10 min. Absorbance of hemoglobin in the supernatant was measured at 545 nm, with 630 nm as reference, in a microplate spectrophotometer (Bio-Rad, iMark, USA). The assay was repeated thrice and the percentage of hemolysis was calculated using the