Apopnexin fitc apoptosis detection kit

The ApopNexin™ FITC Apoptosis Detection Kit (Millipore, Billerica, MA) was used for the annexin V/propidium iodide (PI) flow cytometry analysis of early apoptosis by FACSCaliber cytometry (BD Biosciences) at the Creighton University Flow Cytometry Core Facility. PARP antibody (#9532, Cell Signaling Technology, Beverly, MA) was used to detect cleavage of PARP for the western blot method of apoptosis detection. The percentage of cleaved PARP (89 kDa) in total PARP (116 kDa full length plus cleaved PARP) was used as indicator of apoptosis.

The ApopNexin™ FITC Apoptosis Detection Kit (Millipore, Lake Placid, NY, USA) was performed to quantitate apoptotic cells, according to the manufacturer’s instructions. Briefly, cells were treated with UV irradiation (20 J/m²) followed by 6 h incubation. After a 5-min wash with PBS, 150 μl of an Annexin V antibody in binding buffer was added with incubation for 15 min at room temperature, followed by addition of 1.5 μl of PI at 1 mg/ml and a further incubation for 5 min. Subsequently, after washing with the Annexin V Binding Buffer, samples were immediately analyzed under a fluorescence microscope equipped with a filter for fluorescein isothiocyanate (excitation, 490 nm; emission, 525 nm), and PI staining was assessed with the filter for Texas red (excitation, 570 nm; emission, 610 nm).

Serums and antibiotics were purchased from GIBCO (Grand Island, NY, USA). The 17β-estradiol benzoate [(Estr-1,3,5(10)-trien-3,16α,17β-triol), purity ≥ 98%] was obtained from Sigma-Aldrich (Hamburg, Germany). The 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane sulfonic acid (HEPES, purity ≥ 99.5%) was purchased from Ludwig Biotecnologia Ltd. (Alvorada, RS, Brazil). DMBA (purity ≥ 95%) was purchased from Sigma-Aldrich (Stanford, Germany). Trypan blue (0.4%), Alamar blue, acridine orange, ethidium bromide and cell culture media were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tamoxifen citrate (Mylan^®) was purchased from MYLAN SAS (Saint-Priest, France). The JC-1 probe (5,5′,6′,6-tetrachloro-1,1′,3,3′-tetraethyl benzymidazol carbocianyne iodide) and DCFH-DA (2′,7′-dichlorofluorescein diacetate) were from Invitrogen (Carlsbad, CA, USA). Millicell^® cell culture inserts were purchased from Merck Millipore Ltd. (Tullagreen Carrigtwohill, Ireland). The specific antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The ApopNexin™ FITC Apoptosis Detection Kit was purchased from Millipore (Billerica, MA, USA). Ultrapure Milli-Q water was used to prepare all solutions and buffers in all experiments.

TAMR-MCF-7 cells were plated in 6 well plates and transfected with 120 pmole/well mimic hsa-miR-146b-3p or mimic negative control. The transfected cells were incubated in the presence or absence of 3 μM 4-hydroxytamoxifen in serum free medium for 36 h. The cells were harvested with trypsin and stained with both annexin V-fluorescein isothiocyanate (FITC) and propidium iodide according to ApopNexin FITC apoptosis detection Kit (Millipore, Temecula, CA), and analyzed by flow cytometry (FACStar, BDBiosciences, Mississauga, ON) set for FLH-1 (annexin V) and FLH-2 (propidium iodide). Total 10⁴ cells were counted for each sample.

An ApopNexin FITC Apoptosis Detection Kit (Millipore, Lake Placid, NY, USA) was used to examine apoptotic cells according to the manufacturer’s instructions. A total of 3 × 10⁵ cells were seeded in triplicate in 6-well plates. After 24 h, all cells were incubation with cisplatin, followed by washes with PBS and then the Annexin-V binding solution. Subsequently, 150 μL of the Annexin-V antibody in Binding Buffer was added to each culture well. Cells were incubated for 15 min, 1.5 μL of PI was added at 1 mg/mL, and cells were further incubated for 5 min. A total of 10,000 cells were analyzed on a flow cytometer (FACSCalibur; BD Biosciences).

Cells transiently transfected with siRNA or miRNA were plated in 6-well plates with or without siRNA-100 transfected, then the PV at different titers was added at 24h post-trasfection. After post-infection for 24 h, the cells’ apoptosis was measured by ApopNexin^™ FITC Apoptosis Detection Kit (APT750, Millipore, Temecula, CA) as we previously described44 (link). Briefly, cells were harvested and spun down (400 g, 5 min) and washed by PBS, resuspending cells in 1× bind buffer at a concentration of 10⁶ cells/ml. Then, took 200 μl cell suspension and added 3 μl of the annexin conjugate ApopNexin™ FITC and 2 μl PI. Finally, samples were mixed and incubated for 15 min at room temperature in the dark and placed the cells on ice. Fluorescence due to FITC and PI staining was measured in a flow cytometer (Cytomics FC 500, Beckman Coulter).