br Cell growth analysis br The cell
2.5. Cell growth analysis
The cell growth analysis of MCF-7 and MDA MB-231 Midostaurin (PKC412) was de-termined by trypan blue dye exclusion method as described previously (Koppikar et al., 2010; Choudhari et al., 2013). Briefly, the cells were seeded at a density of 2 × 104 cells/well in 24-well plate in triplicates and were incubated for 24 h at 37 °C. MCF-7 cells were treated with 0–40 μg/ml concentrations and MDA MB-231 cells were treated with 0–80 μg/ml concentrations of HC9 for 24, 48 and 72 h. The cells were harvested and counted for viability by using trypan blue dye.
2.6. Colony formation assay
Clonogenic survival determination assay was performed as ex-plained previously (Koppikar et al., 2010; Choudhari et al., 2013). Briefly, MCF-7 and MDA-MB-231 cells were trypsinized and plated at a density of 1 × 103 cells/well in 6-well plates and incubated for 24 h at 37 °C. The medium was removed and fresh medium was added with the diﬀerent concentrations of HC9 (0–40 μg/ml for MCF-7 and 0–80 μg/ml for MDA MB-231) followed by incubation for 7–10 days at 37 °C. The plates were observed under inverted compound microscope until suf-ficient colonies were formed. Once the colonies were formed, the media was removed and the cells were fixed with 4% paraformaldehyde
S. Suryavanshi, et al.
followed by staining with 0.5% crystal violet dye. The colonies were photographed with Sony DSC-S75 cyber-shot camera.
The assay was performed as described previously (Koppikar et al., 2010; Choudhari et al., 2013). Briefly, MCF-7 and MDA-MB-231 cells (5 × 103 cells/well) along with diﬀerent concentrations of HC9 (0–40 μg/ml for MCF-7 and 0–80 μg/ml for MDA MB-231) were mixed with 0.35% agarose (DNA grade, GIBCO BRL) in complete DMEM medium at 40 °C and gelled at room temperature for 20 min over a previously gelled layer of 0.5% agarose in complete medium in 6-well plates. After incubation for two weeks, the colonies were counted in 10 diﬀerent fields by using an Axiovert 200 M microscope (Carl Zeiss, Germany) and the average value was calculated.
2.8. Cell cycle analysis
Cell cycle distribution was determined by using flow cytometry analysis. Briefly, MCF-7 and MDA MB-231 cells were seeded at a density of 4 × 105 cells/well in 6 well plates and incubated overnight at 37 °C. The cells were treated with HC9 extract (0–40 μg/ml for MCF-7 and 0–80 μg/ml for MDA MB-231) for 24 h. The cells were harvested by trypsinization and fixed in ice-cold 70% ethanol at −20 °C for 30 min. Following washing with 1 × PBS, the cells were treated with RNAse A (50 μg/ml) at room temperature for 30 min and stained with PI (20 μg/ ml). Stained cells were analyzed for DNA-PI fluorescence by using a flowcytometer (FACS Calibur, BD). A minimum of 10,000 events were counted per sample; data were analyzed by using FACS Calibur-cell quest software (Becton Dickinson) for the proportions of cells in G0/G1, S phase and G2/M phases of the cell cycle.
2.9. Wound healing assay
MCF-7 and MDA MB-231 were seeded into 24-well plates at a density of 4 × 105 cells/well and were allowed to adhere overnight at 37 °C in 5% CO2 incubator. After 6 h serum starvation of cells, an ar-tificial wound was made in the plates by using a sterile 10 μl micro-pipette tip and the cellular debris was gently washed with 1XPBS. The wounded monolayer of MCF-7 was treated with 0–40 μg/ml con-centrations and MDA MB-231 cells were treated with 0–80 μg/ml con-centrations of HC9 extract. Migrating cells were examined and the images for 0 h as well as 16 h were captured with the help of Axiovert 200 M microscope. The average extent of wound closure was evaluated by measuring the width of the wound by using ImageJ 1.44p.