Annexin 5 apc

Proliferation was measured in quadruplicate by MTT Cell Proliferation Assay (ATCC) following the manufacturer’s protocol. Apoptosis was measured by flow cytometry after staining with Annexin V-APC (Life Technologies) and PI (Sigma) in 10 mM HEPES at pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂. For cell cycle analysis, cells were washed once in PBS, fixed in 70% ethanol at 4°C, washed again in PBS and resuspended in 0.1% (v/v) Triton X-100 in PBS, containing 200 μg/ml DNase-free RNase A and 20 μg/ml PI. Samples were incubated at 37°C for 15 min before analysis on a Becton-Dickinson FACScan using FlowJo (Tree Star, Inc).

Apoptotic cell death were measured by staining with Sytox Green and Annexin V–APC (Life Technology, USA) as previously described [13 (link)].

Purified EVs were counted using TruCount tubes (BD Biosciences) on an LSRII flow cytometer. HUVECs were incubated with 1 million purified EVs in standard vesicle-free endothelial growth medium. For positive controls cells were treated with 10% ethanol (EtOH) (52 ). After 3 h, cell supernatants were mixed with the trypsinized cells to include detached dead cells in the analysis, and cells were stained with annexin V-APC (A35110; Life Technologies) and PI (421301; BioLegend) in annexin V binding buffer (V13246; Life Technologies). Cells were analyzed by flow cytometry immediately after treatment and staining.

Phosphatidylserine exposure was used to determine cell viability. Cells were washed with PBS, pelleted, and then incubated with annexin binding buffer containing Annexin V-APC (Life Technologies) for 15 min in the dark. Then, samples were analyzed by flow cytometry using FACSCanto II and FACSDiva (BD Biosciences, Franklin Lakes, NJ, USA). Data represent the percentage of non-apoptotic cells (Annexin V-).
Mitochondrial superoxide detection was analyzed with a MitoSOX™ indicator (From ThermoFisher). Jurkat, U2OS, and HEK293T cells (0.2–0.5 × 10⁶) were collected and incubated with MitoSOX™ 15 min prior to the start of the measurement. After that, cells were washed once with 1 mL of complete PBS (c-PBS, PBS supplemented with 0.5 mM MgCl₂, 0.7 mM CaCl₂, and 1 g/L glucose). Fluorescence was detected by flow cytometry with FACSCanto equipment and analyzed using FlowJo software.

T cells were transferred to 96-well round bottom plates (Denville, South Plainfield, NJ) at a density of 5 × 10⁴ T cells/well (sorted T cells) or 2 × 10⁵ splenocytes/well (following transplantation) and treated with increasing concentrations of venetoclax. Cells were cultured in media alone (cDMEM, cytokine deprivation), 1 μg/mL ionomycin, 4 ng/mL PMA, 1 μM etoposide, 250 nM SAHA/vorinostat, and 50 nM staurosporin. Following 24 h incubation, cell death was assessed using Annexin V-APC (Life Technologies) and propidium iodide (Life Technologies) as previously reported [18 (link)].

Hepatocytes from mice were isolated based on standard method as published earlier [28 (link)]. Hepatocytes were treated with one ROS component hydrogen peroxide (HP) for 24 hours at 5mM. EGCG was added at 10μM concentration. Cells were analyzed by flow cytometry for early apoptotic marker Annexin V APC and cell death dye Sytox green (Life Technologies, USA).

The transduced U87 and U251 cells overexpressing control vector or canstatin were trypsinized with 0.25% trypsin-EDTA (Life Technologies) and washed once in PBS with 2% FBS. Cells were then collected and stained with 10 μL Annexin V-APC (Life Technologies). After incubation in the dark for 10 min at room temperature, stained cells were analyzed on a flow cytometer (Beckman Coulter, Inc., Fullerton, CA, USA). ModFit program (Verity Software House Inc., USA) was used to analyze the proportion of apoptotic cells.