Annexin 5 fitc pi staining kit

In order to analyze DNA fragmentation, NCI-H460 and HepG2 cells were induced apoptosis by treating with AB2 at IC₅₀ and 2 × IC₅₀. DNA purification kit was applied to extract DNA, according to the manufacturer’s instructions (Thermo Fisher Scientific, CA, USA). Passing quantification, 2 μg of each DNA sample was loaded to electrophoresis on a 1.5% agarose gel, then the gel was photographed under ultraviolet illumination after staining with ethidium bromide (10 μg/mL).
The effect of AB2 on cell apoptosis was evaluated by flow cytometer with Annexin V-FITC/PI staining kit (Thermo Fisher Scientific, CA, USA), according to the manufacturer’s instructions. Briefly, the cells were treated with AB2 compound at IC50 and 2 × IC50 concentrations for 24 h. After harvesting, cells were suspended in 300 μL binding buffer, and then stained with Annexin-FITC and/or propium iodide. Positive controls for apoptosis were stained with only Annexin-FITC. Positive controls for necrosis were stained with only propium iodide. At least 10⁴ cells were analyzed by flow cytometer (BD, Biosciences, San Jose, CA, USA) and data were analyzed using FlowJo vX.0.7 (Tree Star, Inc., Ashland, OR, USA).

Parthenolide was purchased from MedChemExpress (cat# HY-N0141) and dissolved in DMSO. The Annexin V-FITC/PI staining kit was was purchased from Thermofisher (cat#88-8005-72). The antibodies for western blot are as followings: primary antibodies against phospho-EGFR (cat#3777), total-EGFR (cat#4267), phospho-ERK (cat#4377), total-ERK (cat#4695), phospho-AKT(T473) (cat#4060), phospho-AKT(T308) (cat#13,038), AKT (cat#9272), cleaved-caspase-3 (cat#9661), PARP (cat#9532), Ki-67(cat#9027), HRP goat anti-rabbit (cat#7074) and-mouse secondary antibodies (cat#5127) and GAPDH (cat#5174) were purchased from CST Cell Signaling Technology.

Cell apoptosis was evaluated by flow cytometry using Annexin V-FITC/PI staining kit (Invitrogen, USA). After harvesting, the LoVo cells were trypsinized, rinsed by cold PBS buffer and stained with 5 µL Annexin V-FITC and 10 µL PI solution. Subsequently, the cell apoptosis rate was quantitatively assessed using FACScan flow cytometer (Beckman Coulter, USA).

Apoptosis of cells was detected using an Annexin V–FITC/propidium iodide (PI) staining assay. Flow cytometric analysis of apoptotic cells was carried out using an Annexin V–FITC/PI staining kit (Invitrogen). After washes with cold PBS, the cells were re-suspended in binding buffer (100 mM HEPES, 100 mM NaCl, and 25 mM CaCl₂, pH 7.4) and stained with Annexin V-FITC/PI at room temperature in darkness for 15 min. Apoptotic cells were then evaluated by gating PI and Annexin V–positive cells on an FACSCalibur (BD Biosciences). All experiments were performed in triplicate.

Antibodies against Bcl-2, BAX, PARP, caspase-3, CHOP, Ubiquitin, eIF2α, and p-eIF2α were purchased from Cell Signaling Technology (Beverly, MA, USA). HRD1 antibody for western blot analysis and eIF2α antibody for immunofluorescence staining were obtained from Abcam (Cambridge, MA, USA). HRD1 antibody for immunohistochemistry (IHC) was purchased from Abgent (San Diego, CA, USA). Antibodies against β-actin, tubulin, and GAPDH were acquired from Proteintech (Chicago, IL, USA). The annexin V-FITC/PI staining kit was purchased from Invitrogen (Grand Island, NY, USA). The HA-Ub construct was the gift from Dr. Fei Sun (Wayne State University). Enhanced green fluorescent protein (EGFP)-tagged HRD1 adenovirus (Ad-HRD1) and Myc-eIF2α were obtained from Genechem (Shanghai, China).

The apoptotic profiles of cell and tissue samples were measured using TUNEL and flow cytometry assays, respectively. For flow cytometry assay, an Annexin V‐FITC/PI staining kit (Invitrogen) was used according the kit instructions. For TUNEL assay, a TUNEL kit (Thermo Fisher) was used.

Cell apoptosis of HCT15 and SW116 was monitored by using Annexin V-FITC/PI staining kit (Invitrogen), as guided by supplier. 1 × 10⁶ cells were prepared in 6-well plates for 15 min of double-staining in the dark room. Results were examined by a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).