Annexin 5 fitc propidium iodide

After treatments for 24 h, PANC-1 cells were collected and washed twice with PBS. The cells were then transferred to 100 μl of binding buffer and incubated with an Annexin V-FITC/Propidium Iodide (BD Biosciences, San Jose, CA, USA) staining solution for 15 min at room temperature in the dark, and then added with 400 μl of binding buffer into each tube for final flow cytometry measurement. Statistical analysis of fluorescence was recorded by FACSCanto™ II system (BD Biosciences).

Human Jurkat T lymphocytes, HeLa epithelial cells, and murine thymocytes were exposed to UV irradiation at 254 nm for 10 min followed by incubation in RPMI-1640 with 10% fetal bovine serum for 2 h at 37°C and 5% CO₂. Human neutrophils (>95% purity) were either cultured overnight at 9 × 10⁵/mL in RPMI 1640 at 37°C in 5% CO₂ or UV-irradiated for 10 min followed by incubation for 2 h before addition to macrophages (3 × 10⁵/mL). Evaluation of nuclear morphology using light microscopy on Wright-Giemsa stained samples indicated that the irradiated cells were approximately 70–80% apoptotic [14 (link)]. Apoptosis was confirmed by annexin V-FITC/propidium iodide (BD Biosciences, San Jose, CA) staining followed by flow cytometry analysis on a FACSCalibur system (BD Biosciences) [15 (link)]. Human aged neutrophils were shown to be typically 60–70% apoptotic by assessment of nuclear condensation on Wright-Giemsa stained samples and necrosis was less than 2% by trypan blue exclusion [16 (link)].

Cells were cultured in 35 mm culture plates for 24 hours before being treated with medication. Flow cytometry was utilized for analyzing the cells using Annexin V-FITC/propidium iodide (PI) or JC-1 green/JC-1 red detection kits (BD Biosciences, Franklin Lakes, NJ). Before collecting cells in PBS, they were washed twice with phosphate-buffered saline (PBS). Cells were incubated for 15 minutes at room temperature in the dark in 100 μL of binding buffer. The samples were automatically acquired using the loader with 10,000 event detection criteria for each tube, and the quadrants were constructed based on the viable population. The FlowJo v10.6 software was used to evaluate the results.

Anti-Caspase-3, Anti-Caspase-8, Anti-Caspase-9, and anti-PARP antibodies for Western blotting were purchased from Cell Signaling Technology (Danvers, MA). Anti-Calreticulin antibody was purchased from Enzo Life Sciences (Farmingdale, NY). Anti-rabbit and anti-mouse HRP conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). For cell viability assays, Annexin V FITC/propidium iodide (BD Biosciences) was used following the protocol of the manufacturer.

AML cells were treated with increasing doses of AHCC (0, 1, 5, 10 mg/ml) for 24 or 48 hours. Cells were either subjected to Trypan Blue (Sigma St. Louis, MO) or harvested and stained with Annexin V FITC/propidium iodide (BD Biosciences) using the protocol of the manufacturer.

Human Jurkat T lymphocytes, human HeLa epithelial cells, and murine thymocytes were exposed to ultraviolet irradiation at 254 nm for 10 min followed by incubation in RPMI-1640 with 10% FBS for 2 h at 37°C and 5% CO_2. Evaluation of nuclear morphology using light microscopy on Wright-Giemsa-stained samples indicated that the irradiated cells were approximately 70–80% apoptotic [21 (link)]. Lysed (necrotic) Jurkat T cells were obtained by multiple freeze-thaw cycles [56 (link)]. Apoptosis and necrosis were confirmed by Annexin V-FITC/propidium iodide (BD Biosciences, San Jose, CA) staining followed by flow cytometric analysis on a FACSCalibur system (BD Biosciences) [8 (link)].