Annexin 5 fitc assay

Analysis of cellular apoptosis of untreated and treated HepG2 cells was investigated using Annexin V-FITC assay (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. This procedure was performed in 6 mL polystyrene round-bottom FACS tubes. The optimum concentration was 2 to 4 × 10⁶ cells per 200 μL volume for flow cytometry analysis. Cells were treated with the IC₅₀ of CADMN at different time points (24, 48 and 72 h). The experiment was repeated three times for each time point. The cells were washed two times with cold PBS, then Annexin binding buffer (1×) was added at the concentration of 1 × 10⁶ cells/mL. Then 5 µL Alexa Fluor Annexin V conjugate and PI (5 µg/mL) was added to (100 µL) cell suspension and incubated in the dark for 30 min. After that, binding buffer (400 µL) was added to each tube and analyzed by FACSCalibur flow cytometer system (Becton Dickinson, San Jose, CA, USA) [12 (link)].

Approximately 7.5 × 10⁵ cells were plated in 100 mm plates. After overnight incubation, cells were treated with 350 μg/mL of AGS extract for 24 + 24 h and stained with Propidium Iodide and Annexin V (Annexin V FITC assay, BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer’s guidelines. Flow cytometry was performed using a flow cytometry system (FACSCanto, BD Biosciences Franklin Lakes, NJ, USA). All experiments were performed in biological triplicate and data are expressed as the mean ± SD.

To evaluate the effect of arsenic trioxide on early apoptosis, the Annexin V-FITC assay (BD Pharmingen) was performed according to a previous described protocol [30 (link)–33 (link)]. Briefly, A549 cells were seeded at a density of 3 × 10⁵ cells in F12-K complete medium on 13 × 100 mm tissue treated plates, and grown to 60–70% confluence. Cells were serum starved overnight in 1% FBS in F12-K medium supplemented with 1% penicillin/streptomycin. The serum medium was removed. The cells were reintroduced to F12-K complete medium, and treated with ATO at 0, 2, 4, and 6 µg/ml for 48 hr. The cells were washed twice with cold PBS and then resuspended in 1× binding buffer [10 mM Hepes/NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl₂]. The cells (100 µL) were transferred to a 5ml culture tube and 5 µL of Annexin V-FITC and 5 µL propidium iodide. The cells were vortexed and incubated for 15 min at room temperature (25°C) in the dark. Four hundred micro-liters of binding buffer (1×) was added to the tubes, analyzed and counted at 10,000 counts using a fluorescence-activated cell-sorting (FACS-Vantage) system (Becton-Dickinson, San Jose, CA).

Cell viability of MM cells was determined by using MTS (3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium) assay [7 ]. Rate of apoptosis was checked by using Annexin-V-FITC assay (BD Pharmingen, San Jose, CA, USA) according to manufacturer's protocol. Percentage specific apoptosis was calculated as previously described [7 ]. Protein levels of cleaved-PARP and cleaved-caspases-8, -9 and -3 were used as a semi-quantitative gauge for apoptosis.

To perform Annexin V assay on the Panc 03.27 cell lines, Annexin V FITC Assay (BD Biosciences) was used. Following esiRNA transfection and/or 5-FU treatment, cell lines were detached using Accutase, and spun at 300 rpm for 5 minutes together with used medium to collect released/dead cells. Medium was removed, and Annexin V was added to cells according to product protocol. Acquisition of data was performed using an EasyCyte flow cytometer (GuavaTechnologies). Exclusion of non-viable cells and debris was based on lower forward scatter and side scatter properties. Data analysis was performed using Flowing Software (Version 2.5.1).

The Annexin V-FITC assay (556547, BD, USA) was used to evaluate cell apoptosis. DPCs were incubated with 5 μM TPEN for 24 h. Then 5 μg mL^-1 ZnMOF solution, BMN extract, or ZnMOF-MN extract were added in corresponding groups. DPCs cultured in DMEM/F-12 (0.1% DMSO) without TPEN were used as the control group. After 24 h of treatment, cells were collected by using Trypsin (0.25%) and washed twice with PBS. Next, 5 μL Annexin V-FITC, 5 μL of PI and 500 μL of binding buffer were added to each group and gently mixed up. After 15 min of incubation, apoptosis was detected with a flow cytometer (Beckman Coulter, USA). The sum of the cell proportion in upper right (UR) and lower right (LR) represents the total apoptosis rate.