Annexin 5 alexa fluor 488 pi

The measurement of cell death was performed by the guidelines of (35 (link)). Detection of apoptotic/necrotic cells was determined by flow cytometry using the Alexa Fluor^® 488 Annexin V/PI (Thermo Fischer Scientific), and the analyses were performed according to the manufacturer’s instructions. Briefly, DU-145 cells were seeded (5.0 × 10⁴ cells/well) in 12-well plates and maintained to attach at 37°C in 5% CO₂ for 24 h. After, cells were treated with 0.25, 0.5, 1.0, and 1.5 μM of both complexes. Cisplatin (Fauldcispla^®) and doxorubicin hydrochloride (Fauldoxo^®) were used as the reference cytotoxic drugs. DMSO (0.1% v/v) was used as the vehicle control. Following 24 h of incubation, the cells were collected with Accutase^® (Gibco-Invitrogen^®), washed with PBS and resuspended in 200 μl of cold annexin-binding buffer. Next, 10 μl of Alexa Fluor^® was added to Annexin V (50 μl/ml) staining buffer, and the mixture was incubated in the dark at 4°C for 15 min. Previously, the analyses, 100 μl of PI (2 μg/ml) was added, and then, the fluorescence was measured by flow cytometry in a FACSCanto flow cytometer (BD Biosciences, USA) using the Diva software. Ten thousand events were evaluated per experiment, and cellular debris was omitted from the analysis.

Cell viability was determined by a fluorescence cell counter (Luna, Logos Biosystems, South Korea) using acridine orange/propidium iodide (PI) staining and a flow cytometric using annexin/PI staining (Alexa Fluor 488 annexin V/PI, Thermo Fisher, MA, USA) according to the manufacturer's protocol.

Cells were seeded at a concentration of 1 × 10⁵ cells per a 60 mm plate and treated with KC-53 or Doxorubicin (Dox) as indicated. Cells were harvested and stained using Annexin-V Alexa Fluor® 488/PI, as described by the Tali™ apoptosis kit (Life Technologies, Carlsbad, CA). Cell viability, death and apoptosis were evaluated using the Tali™ Image-based Cytometer (Life Technologies, Carlsbad, CA). The Annexin-V positive/PI negative cells were recognized as early apoptotic cells by the cytometer software whereas the Annexin-V positive/PI positive cells were identified as late apoptotic/dead cells.

Cells were plated in 24-well plates (5 × 10⁴ cells/well) and treated with FX (25 μM), FxOH (1, 5 or 10 μM), SM-7368 (20 μM) and in combination. Cells were harvested 12, 24, 36 and 48 h posttreatment and stained using annexin-V Alexa Fluor® 488/PI (propidium iodide), as described by the Tali® Apoptosis Kit – Annexin V Alexa Fluor® 488 and propidium iodide (Life Technologies Corporation, Van Allen Way Carlsbad, CA, USA). Cell viability, death and apoptosis were evaluated using the Tali® Image-based Cytometer (Life Technologies Corporation). The annexin-V positive/PI-negative cells were recognized as early apoptotic cells by the cytometer, whereas the annexin V-positive/PI-positive cells were identified as late apoptotic cells. Similarly, the annexin V-negative/PI-negative cells were identified as viable cells and the annexin V-negative/PI-positive cells were identified as dead cells.

HCT116 cells were seeded in six-well plates at a density of 1 × 104 cells per well and incubated at 37°C until a confluence of 80%. The cells were treated with PTX, Rev-PTX NPs, LPK-PTX NPs at normalized PTX concentration of 5 μg/ml at 37°C for 48 h. Then the cells were trypsinzed, washed, and stained with Annexin V-Alexa Fluor488/PI according to manufacturer’s protocol (Life Technologies, United States). The cell apoptosis was monitored by flow cytometry (BD, CA, United States) and the apoptotic percentage of three independent experiments was analyzed.