Annexin 5 pi double staining assay kit

For the detection of cell death we used simple propidium iodide (PI, Biotium, Fremont, CA, 40016) uptake assays (as in^{61 (link)}), while to differentiate between apoptosis and necrosis we used an Annexin V + PI double staining assay kit (Invitrogen, Oregon, USA, V13242).

IS induced cytotoxicity was assessed by simple propidium iodide (PI; Biotium, Fremont, CA, 40016, USA) uptake assays, as described in Kovacs et al. [17 (link)]. Cells were seeded in six-well plates (4T1, 75,000 cells/well; MCF7, 150,000 cells/well; SKBR-3, 200,000 cells/well; human fibroblasts, 200,000 cells/well) and treated with the indicated concentrations of IS for 24 h followed by staining with 100 μg/mL PI for 30 min at 37 °C. Adherent cells and supernatants were collected in FACS tubes, washed once with PBS, and analyzed by flow cytometry (FACS Calibur, BD Biosciences).
To evaluate changes in necrotic and apoptotic cell death, we used an Annexin V+PI double staining assay kit (Invitrogen, Carlsbad, CA, USA, V13242). Cells were seeded in six-well plates (4T1, 75,000 cells/well; MCF7, 150,000 cells/well; SKBR-3, 200,000 cells/well; human fibroblast, 200,000 cells/well) and treated with the indicated IS concentrations for 24 h. Then, the collected cells were stained with 100 μg/mL PI solution and 5 μL FITC Annexin V, according to the manufacturer’s instructions. The numbers of apoptotic and necrotic cells were measured using a FACS Calibur flow cytometer.

IPA-induced cytotoxicity was assessed by propidium iodide (PI; Biotium, Fremont, CA, 40016, USA) uptake assays as in [55 (link)]. Cells were seeded in a 6-well plate (4T1—75,000 cells/well; MCF7—150,000 cells/well; SKBR-3—200,000 cells/well; human fibroblast—200,000 cells/well) and treated with the indicated concentrations of IPA for 24 h; then, they were stained with 100 μg/mL PI for 30 min at 37 °C, washed once in PBS, and analyzed by flow cytometry (FACS Calibur, BD Biosciences).
To assess changes in apoptotic and necrotic cell death, we used an Annexin V+PI double staining assay kit (Invitrogen, OR, USA, V13242). Cells were seeded in 6-well plates (4T1—75,000 cells/well; MCF7—150,000 cells/well; SKBR-3—200,000 cells/well; human fibroblast—200,000 cells/well) treated with the indicated IPA concentrations for 24 h. Then, cells were stained with 100 μg/mL PI solution and 5 μL FITC Annexin V according to the manufacturer’s instructions. The number of apoptotic and necrotic cells were counted using a FacsCalibur flow cytometer (Beckton-Dickinson Franklin Lakes, NJ, USA).