Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit

Cell apoptotic rate was examined via flow cytometry (Agilent, Santa Clara, CA, USA) using an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Briefly, 2 × 10⁵ T24 and 5637 cells were inoculated into 12-well plates. After 72 h of culture, cells were harvested and resuspended in Annexin V binding buffer. Next, cells were dyed with Annexin V-FITC and propidium iodide (PI) for 10 min. Cell apoptotic patterns were analyzed using a flow cytometry. Cell apoptotic rate was represented as the percentage of cells at early (Annexin V-FITC⁺ and PI⁻) and late (Annexin V-FITC⁺ and PI⁺) apoptotic phases.

The same numbers of LLC cells (2×10⁵/well) were seeded onto 24-well plates and incubated in culture medium from RAW 264.7 cells treated with the indicated concentration of HAD-B for 24 h. Apoptosis was measured using the Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Thermo Fisher Scientific, Inc.). The control and HAD-B-treated cells (1×10⁶) were re-suspended in 500 µl of binding buffer and incubated with 5 µl of Annexin V-FITC and 1 µl of propidium iodide (PI) solution for 15 min. The samples were then examined on a BD FACSCanto II (BD Biosciences, Franklin Lakes, NJ, USA), measuring excitation/emission at 494/525 nm for Annexin V-FITC and 535/617 nm for PI.

Dulbecco's modified Eagle's medium (DMEM; cat. no. 11965118) and fetal bovine serum (FBS; cat. no. 16140071) were supplied by Gibco; Thermo Fisher Scientific, Inc. Annexin V/fluorescein isothiocyanate (FITC) apoptosis detection kit (cat. no. 556547) was purchased from BD Biosciences. Cell counting kit-8 (CCK-8; cat. no. C0038), JC-1 mitochondrial membrane potential detection kit (cat. no. C2006) and lactate dehydrogenase (LDH) cytotoxicity assay kit (cat. no. C0016) were purchased from Beyotime Institute of Biotechnology. 2', 7'-dichlorofluorescein acetyl acetate (DCFH-DA; cat. no. D6883) kit was supplied by Sigma-Aldrich; Merck KGaA. Rabbit polyclonal antibodies against B-cell lymphoma 2 (Bcl-2; cat. no. ab32124) and Bcl-2-associated X protein (Bax; cat. no. ab32503) were purchased from Abcam. Rabbit monoclonal antibody against NAPDH oxidase 2 (NOX2; cat. no. ALX-350-100-C050) was purchased from Enzo Life Sciences, Inc. Rabbit monoclonal antibodies against histone H3 (cat. no. 7631), Keap 1 (cat. no. 8047), AMPKα (cat. no. 5832), phospho (p)-AMPKα (Ser485; cat. no. 2537), Nrf2 (cat. no. 12721) and GAPDH (cat. no. 5174) were obtained from Cell Signaling Technology, Inc.

Apoptotic cells were examined using the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (Thermo Fisher Scientific, Inc.). Cells (3×10⁵ cells/well) were seeded into a six-well plate and incubated overnight at 37°C. The next day, cells were treated with various concentrations of AG (20, 50, and 75 µM) for 24 h and resuspended in 500 µl of binding buffer (1X) to obtain a cell density of 2×10⁵ cells/ml. The cells were then incubated with 5 µl of Annexin V-FITC and 10 µl propidium iodide (PI; 20 µg/ml) in cell suspension for 15 min at room temperature. Fluorescence intensities of the samples were determined using the FACS Canto II flow cytometer (BD Biosciences). In the histogram of FACS analysis, apoptotic cells were represented the combination of Q2 (PI⁺/Annexin V⁺, late apoptotic cell) and Q3 (PI⁻/Annexin V⁺, early apoptotic cell), and dead cells were represented the combination of Q1 (PI⁺/Annexin V⁻, necrotic cell) and Q2 (PI⁺/Annexin V⁺, late apoptotic cell).