Annexin 5 fitc

Cell cycle and cell death induction after MCU silencing were measured by cytofluorometry. Apoptotic and necrotic cells were identified by labeling with FITC‐Annexin V (Roche) and propidium iodide (Sigma) for 15 min at 37°C and analyzing cells by FACS (FACS Canto II, BD BioSciences). Data were processed using the BD Vista software.

To examine the effect of tumor cells on lymphocyte apoptosis, 5×10⁶ MDA-MB-231 SCR cells and KD cells were cocultured with 5×10⁵ Jurkat leukemia T cells in the presence of mouse IgG (2 or 5 μg/ml; 17314; IBL Co., Ltd., Gunma, Japan), anti-human PD-L1 antibody (5 μg/ml; MIH1; BD) or anti-PD-1 antibody (2 μg/ml; EH12.2H7; BioLegend) in a 6-well plate for 24 hours. To block the PD-1/PD-L1 pathway, SCR cells were preincubated with anti-human PD-L1 antibody (5 μg/ml; BD) for 30 min or Jurkat cells were preincubated with anti-PD-1 antibody (2 μg/ml; BioLegend) for 30 min. Extent of apoptosis in Jurkat cells was determined by flow cytometry using FITC-Annexin V (11858777001; Roche) according to the manufacturer's instructions.

The effect of miR-154 on cell apoptosis was assessed by employing a flow-cytometric detection kit containing FITC-Annexin V and propidium iodide (PI) (Roche Applied Science, Germany) following the instructions provided by the kit. A total of 3 × 10⁵ of MCF-7 and MDA-MB-231 cells were seeded in 6-well plates and after 48 h, cells were harvested and after washing twice with PBS, were stained with Annexin V as well as PI. Finally, evaluation of stained cells was done by flow-cytometry (FACScan, BD Biosciences, USA) equipped with bandpass filters at 515 nm for detection of FITC, 600 nm for PI detection and 488 nm laser for excitation.
CellQuest software (BD Biosciences) was employed for estimation of obtained results. The cells that were positive for Annexin V-FITC were presented as cells that had undergone apoptosis.

Neutrophil apoptosis and loss of plasma membrane integrity were analyzed by flow cytometry (FACSCalibur, BD Biosciences, Oxford, UK or Attune NxT, Thermo Fisher Scientific, Loughborough, UK) of FITC-annexin V (Roche, Welwyn Garden City, UK) and propidium iodide-stained neutrophils^{18 (link)}. Activated caspase-3 was measured using an AF488-coupled antibody specific for cleaved human caspase-3 according to the manufacturer’s instructions. Data were analyzed using FlowJo (TreeStar; version 6.4.7) or FCS Express 7 (De Novo).

To quantify apoptosis, cells were harvested and stained with annexin V-FITC and propidium iodide (Roche, United States). The apoptotic cells (annexin V-positive and PI-negative) were analyzed using a FACSCalibur flow cytometer (BD Biosciences, United States).