Apc annexin 5 apoptosis detection kit with propidium iodide

For detection of apoptosis and necrosis, the APC Annexin V Apoptosis Detection Kit with propidium iodide (PI) (BioLegend, San Diego, CA, USA) was used, following the manufacturer’s instructions. The harvested cells were washed twice with PBS and resuspended in binding buffer at a density of 1 × 10⁶ cells/mL. The cells were treated with Annexin V and the PI reagent and incubated for 15 min in the dark. Samples were assayed using an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA).

Apoptosis assay was performed in Huh7 and PLC/PRF/5 cells treated 48 h with DMSO (vehicle control) or with the indicated doses of FH535, Y3, or FH535-M. Cells were harvested and stained with the APC Annexin V apoptosis detection kit with Propidium Iodide (PI) (BioLegend, USA) according to the manufacturing instructions followed by flow cytometry analysis. Flow cytometry data was acquired with an LSRII instrument (BD-Biosciences) and analyzed with FlowJo software (Tree Star).

APC Annexin V Apoptosis Detection
Kit with propidium iodide (PI) (Biolegend, San Diego, USA) was used
to determine cell death. Briefly, U87-MG cells were seeded on a 6-well
plate at a density of 25 × 10⁴ cells per well and
incubated at 37 °C with 5% CO₂ overnight. The cells
were treated with 5 μg/mL of DOX, DOX-loaded micelle (EK255),
and empty micelle (EK257) groups for 2 and 4 h. Cells were harvested
and the pellets were re-suspended in 100 μL of 1× Annexin
V binding buffer. The cells were then incubated with 5 μL of
Annexin V-FITC and 10 μL of PI for 15 min in the dark at room
temperature and 400 μL of 1× Annexin V binding buffer was
added, according to the manufacturer’s instructions. Cell fluorescence
was measured by flow cytometry (NovoCyte, ACEA Biosciences Inc., CA,
USA). The ratio of cell death was assessed with single PI positive
cells (Q2–1). Early apoptosis and late apoptosis were detected
by single APC positive cells (Q2–4) and APC and PI double positive
(Q2–3) cells, respectively.

Apoptosis was evaluated by flow cytometry using BioLegend's APC Annexin V Apoptosis Detection Kit with propidium iodide (PI) (cat. no. 640932 BioLegend, San Diego, CA) following the manufacturer's instructions. Prepared cells were analyzed on the BD FACSAria™ III flow cytometer (BD Biosciences, Bedford, MA, USA) using FACSDiva Software. A total of 10,000 events per condition was recorded. Live cells are negative for both Annexin V APC and PI. Early apoptotic cells are Annexin V APC positive and PI negative. Late apoptotic/necrotic cells are positive for both Annexin V APC and PI.

Apoptosis assays were performed using APC Annexin V Apoptosis Detection Kit with Propidium Iodide (PI) (Biolegend, 640914 San Diego, CA, USA). Briefly, cells were plated at a density of 100,000 cells per dish in 35 mm dishes (Eppendorf 0030 700.112, Hamburg, Germany). Following WPSC treatment, the cells were collected at the indicated timepoints by trypsinization and subsequently washed with 1× PBS prior to labeling with Annexin V-APC and PI following the manufacturer’s protocol. Acquisitions of 20,000 cells were performed using a Biorad S3E Cell Sorter and data processed using the FCS Express flow cytometry program (De Novo Software, Pasadena, CA, USA). Annexin V-positive cells were classified as apoptotic.

Cells were harvested and stained using antibodies for the cell markers CD11b (Biolegend), CD14 (Biolegend) to detect the cell differentiation. The mixture was incubated in 100 μl PBS supplemented with 2% FBS in darkness for suitable length of time at 4 °C before detection. An isotype-matched antibody served as a negative control. Cells were stained with CFSE and cultured for 48–72 h before measuring the intensity of fluorescence to detect cell division (Beyotime). For the apoptosis assay, the cells were stained using the APC Annexin V Apoptosis Detection Kit with Propidium Iodide (PI) (Biolegend) according to the manufacturer's instructions and then detected by flow cytometry analysis. Flow cytometry was performed on eight-laser cytometers (BD). All data were analyzed using FlowJo software.

Apoptosis analysis was performed using the APC Annexin V Apoptosis Detection Kit with propidium iodide (PI) (BioLegend). The analysis was performed based on the manufacturer’s instructions. Briefly, the cells were washed twice with cold BioLegend Cell Staining Buffer and then resuspended in Annexin V Binding Buffer at a concentration of 0.5 × 10 cells/ml. We transferred 100 µl of cell suspension to a new tube and added 5 µl of APC Annexin V and PI solution. The cells were vortexed gently and incubated for 15 min at room temperature (25 °C) in the dark. Finally, 400 µl of Annexin V Binding Buffer was added to each tube and analyzed by flow cytometry using a FACSariaII System (BD Biosciences).