Annexin 5 and dead cell assay kit

The evaluation of the percentage of the apoptotic cells in samples was performed using Annexin-V and Dead Cell Assay Kit from Merck Millipore, Darmstadt, Germany according to supplier’s protocol. Briefly, cells were seeded on the 6-well plastic plates in the complete DMEM and were incubated at 37 °C, in 5% CO₂, in the presence or not of the related treatments. After incubation time, cells were collected using Trypsin–EDTA 1 × in PBS without calcium, magnesium and phenol red, washed with HBSS and centrifuged at 324 g, 5 min, 4 °C and suspended in HBSS with addition of the 1% BSA. The Muse Annexin V and Dead Cell Reagent was added to the cell’s suspension in ratio 1:1 (v/v) and after 20 min incubation at RT in the dark, cells were read out using Muse™ Cell Analyzer equipped with Muse™ Software. The percentage of apoptotic cells was calculated with an assumption that double negative cells are viable cells, only annexin V positive are early apoptotic cells, only 7-AAD positive are necrotic cells and finally double positive cells are late apoptotic cells. Each experiment was performed independently 3 times.

The number of live, apoptotic, and dead cells was evaluated by Annexin V and Dead Cell Assay kit (Merck Millipore, Darmstadt, Germany) and Muse™ Cell Analyzer (Merck Millipore) using 3 × 10⁵ untreated and treated HepG2 cells and a protocol already reported in our recent paper [44 (link)].

The apoptotic effects of the fraction (fraction C) on HeLa and SKOV-3 cells were estimated using Annexin V and Dead Cell Assay Kit (Merck Millipore). The cells were seeded in 12-well plates at a density of 1 × 10⁵ cells/well and treated with the fraction at concentrations of 3.0–100.0 µg/mL. The fraction solvent – methanol was added to the control cells at a concentration of 1.0% (v/v). After treating, the cells were prepared as described previously (Stefanowicz-Hajduk et al. 2016 (link)) and analyzed by flow cytometry with Muse Cell Analyzer (Merck Millipore), following the protocol provided by the manufacturer. The experiments were performed in three independent repeats.

After being treated with CGDCM, 5-FU, or a combination of both for 24 h, cells were harvested and stained with Annexin V and Dead Cell Assay Kit (MCH100105, Merck, Germany), as per the manufacturer’s instructions. Stages of apoptotic cell death were determined by double staining with annexin V and dead cell marker, 7-amino-actinomycin D (7-AAD). Annexin-V stains phosphatidylserine in the early stages of apoptosis, whereas both annexin V and 7-AAD stain cells in the late apoptotic stages. Briefly, cells in 1% FBS phosphate-buffered saline were stained with annexin V and 7-AAD and incubated for 20 min in the dark. Apoptotic cells were analyzed using Muse Cell Analyzer (0500–3115, Merck, Germany).

To study the effect of FTO depletion on apoptosis and the cell cycle, 1 × 10⁵ synchronized MiaPaca2 cells (NTC, shFTOa, and shFTOb) were seeded in the MW 6. After 48 h of cell adherence, the percentage of cells in the G0/G1, S, and G2/M phases of the cell cycle and apoptotic cell fraction were determined using a Cell Cycle Assay Kit and Annexin V and Dead Cell Assay Kit (Millipore, Chicago, IL, USA) on a Muse Cell Analyzer (Millipore) as per the manufacturer’s instructions. In addition, the nuclear blebbing caused due to apoptosis was evaluated by Hoechst staining. Briefly, 1 × 10⁵ cells were seeded on the sterile coverslips in MW 12 and allowed to adhere. Cells were then stained with Hoechst-33258 dye (1 mg/mL) for 20 min and fixed using 4% paraformaldehyde for 15 min. The coverslips were then mounted and observed using a confocal microscope.