Flow cytometry

To analyze cell apoptosis and cell cycle, the KYSE150 and KYSE450 cells were harvested after transfection with siRNA duplexes for 72 h. For cell apoptosis analysis, collected cells were incubated with Annexin V-FITC and propidium iodide (Beyotime, shanghai, China) for 15 min and then detected with flow cytometry (Beckman, USA). For cell cycle analysis, we fixed the collected cells using 70% ethanol overnight at 4 °C. After that, the samples were incubated with propidium iodide (Beyotime, Shanghai, China) at 37 °C for 30 min in the dark. Next, the cells were tested within 2 h by flow cytometry (Beckman, USA).

BxPC-3 and PANC-1 cells were grown into 6-well plates at a density of 5 × 10⁵ cells/well and treated with 5-Aza -2′-deoxycytidine at a concentration of 0 μM or 5 μM for 72 h. Cells were then quantified by flow cytometry using the Annexin V/PI Apoptosis Kit (Multiscience, China). Briefly, cells were washed in cold PBS, re-suspended in 1X binding buffer, and incubated with 5 μl of FITC Annexin V and 10 μl of propidium iodide (PI) for 15 minutes in the dark. The cells were then re-suspended in 400 μl of 1X binding buffer and analyzed immediately by flow cytometry (Beckman Coulter). For cell cycle analysis, the cells were fixed in 70% ethanol and stained with 10 μl Reagent A (Multiscience, China). Then, the cells were sorted by flow cytometry (Beckman Coulter) and cell cycle profiles were analyzed by ModFit software.

After washing with PBS, cells were fixed in 75% ethanol at 4°C overnight. Again washed with PBS, propidium iodide was used to stain cell in the dark at 37°C. Flow Cytometry (Beckman Coulter, Inc., Brea, CA) was used to measure the cell populations in different phases. To determine the cell apoptosis, Annexin V‐FITC Kit (Becton, Dickinson and Company, Franklin Lakes, NJ) was used according to the manufacturer's protocol. Finally, Flow Cytometry (Beckman Coulter, Inc.) was used to measure the cell populations in apoptosis. All assays were carried out biological independently in triplicate.

ROS was measured using an ROS Assay Kit, according to the manufacturer's recommendations. Briefly, cells were loaded in fluorescent probe 2ʹ—7ʹ-dichlorofluorescein diacetate (DCFH-DA) and incubated in a 37 °C cell culture incubator for 20 min. After washing off the excess probes, DCF fluorescence intensity was detected under the fluorescein Isothiocyanate (FITC) channel using flow cytometry (Beckman, IN, USA). ROS in the cell can oxidize non-fluorescent DCFH to produce fluorescent DCF. Detection of the fluorescence of DCF can tell the level of ROS in the cell.
MMP was detected using the MMP Assay Kit with JC-1. The essential assay mechanism can be explained as follows. When the MMP is high, JC-1 aggregates in the matrix of mitochondria to form polymers, which can produce red fluorescence. Whereas, when the MMP is low, JC-1 cannot accumulate in the mitochondrial matrix, and JC-1 is a monomer and produce green fluorescence. Cells were incubated with JC-1 staining working solution at 37 °C for 20 min, and MMP was detected using flow cytometry (Beckman, IN, USA). The JC-1 monomer was measured using the FITC channel, while the JC-1 polymer was detected under the PE channel. The decrease MMP can be easily detected by the transition of JC-1 from red to green fluorescence, and the proportion of mitochondrial depolarization is measured by the relative ratio of red to green fluorescence.

Cell cycle analysis and apoptosis assay were performed as previously described with some modifications (70 (link)). A 50 g/ml propidium iodide (BD Biosciences, USA) was applied to label cells for 30 min in binding buffer. The cell cycle distribution was detected with flow cytometry (Beckman Coulter), which was then analyzed using Modfit software.
The apoptosis was studied with flow cytometry (Beckman Coulter). SKOV-3 cells were labeled by Annexin V-FITC/PI, and then was subjected to be examined. Data were analyzed using FlowJo software.

The cell cycle analysis was performed using flow cytometry (Beckman Coulter, Miami, FL, USA) and PI staining. The ovarian cancer cells treated with 0, 250, 500 and 1,000 μg/ml of PZH were dissociated from the culture plates using a solution of 0.1% trypsin in PBS for 3 min. Next, the OVCAR-3 cells were collected, adjusted to a concentration of 1×10⁶ cells/ml and fixed in 70% ethanol at 4°C overnight. The fixed cells were then washed twice with cold PBS and incubated for 30 min with RNase (8 μg/ml), 0.1% Triton X-100 and PI (10 μg/ml). The fluorescence signal was detected by flow cytometry (Beckman Coulter) through fluorescence channel 2, and the proportion of DNA in each phase was analyzed by Modfit LT version 3.0 (Verity Software House Inc., Topsham, ME, USA).