Annexin 5 fitc apoptosis detection kit

Apoptotic cell death in the HL-60 cells was evaluated by Annexin V and PI staining with a MEBCYTO® Apoptosis Kit (MBL, Nagoya, Japan), while an Annexin V-FITC Apoptosis Detection Kit (Nacalai Tesque) was used for the T98G, MDA-MB-231, and KATO III cells. Fluorescent cells were analyzed using a fluorescence-activated cell sorting (FACS) system (BD FACSAria^TM III, Becton Dickinson, Franklin Lakes, NJ, USA) or a fluorescent microscope (BZ-X710, KEYENCE, Osaka, Japan). Notably, cells were plated in glass-bottom dishes (Matsunami Glass, Osaka, Japan) for this assay.

We seeded A431 cells onto 12-well plates (1.2 × 10⁵ cells per well), transfected them with control siRNA, OVOL1 siRNA, or OVOL2 siRNA; and incubated them for 48 h at 37°C in 5% CO₂. We determined the apoptotic status of the transfected cells using an Annexin V-FITC Apoptosis Detection Kit (15342-54; Nacalai Tesque Inc.) according to the manufacturer’s instructions. Briefly, we harvested the cells by trypsinization and suspended them in 1×Annexin V binding solution at 1.0 × 10⁶ cells/mL. We mixed 100 μL cell suspension with 5 μL Annexin V-FITC solution and 5 μL propidium iodide solution and then incubated them for 15 min at room temperature in the dark. Then, we mixed the cells with 400 μL 1×Annexin V binding solution and analyzed them using a FACSCanto II flow cytometer (BD Biosciences). We determined the proportion of FITC-positive apoptotic cells among live cells by using FlowJo software (663335; BD Biosciences). We conducted all assays in triplicate wells and repeated them at least three times in separate experiments.

To collect dead and detached cells, floating cells in culture supernatants were collected and mixed with cells detached by trypsinization. For DNA and cyclin B1 staining in fixed cells, the cells were fixed with 70% ethanol at −30°C for 1 hour. After washing the cells with PBS(−) containing 3% calf serum plus 0.1% Triton X‐100, the cells were incubated at room temperature with anti‐cyclin B1 antibody and subsequently with Alexa Fluor 488‐labelled donkey anti‐rabbit IgG antibody in PBS(−) containing 3% calf serum plus 0.1% Triton X‐100 for 1 hour in the dark. Then, the cells were stained for DNA with 50 µg/ml propidium iodide (PI) plus 200 µg/mL RNase A at 37°C for 30 minutes in the dark. For DNA and Annexin V staining in unfixed cells, an Annexin V‐FITC Apoptosis Detection Kit (Nacalai Tesque, Kyoto, Japan) was used according to the manufacturer’s instructions. In brief, collected cells were incubated at room temperature with Annexin V‐FITC conjugate and PI in Annexin V binding solution for 15 minutes in the dark. Immediately, the stained cells were analysed using a flow cytometer equipped with a 488 nm solid‐state blue laser and a 640 nm diode red laser (BD Accuri C6 Plus; BD Biosciences, San Jose, CA). Debris was excluded based on the forward and side scatter profiles. FlowJo software (Tree Star, Ashland, OR) was used for data analysis and plot.

C2C12 cells were cultured in 24-well chamber slides and flow cytometry analyses were performed on day 5 with Annexin V-FITC Apoptosis Detection Kit (Nacalai tesque, Kyoto, Japan) according to the manufacturer’s recommendations. FACS Canto II and FlowJo version 10 software were used for obtained data and analyzation.

At 48 h after transfections, chondrocytes cells were seeded into 96‐well plates with 3000 cells in 0.1 ml medium per well. Cells were treated with 10 μg/ml LPS for 48 h in culture media. Then cells were washed with fresh medium, digested by trypsin, and collected by centrifugation. Cell apoptosis was detected using Annexin V‐FITC Apoptosis Detection Kit (15342‐54; nacalai tesque). In brief, cells were resuspended in 195 μl Annexin V‐fluorescein isothiocyanate (FITC) binding solution, followed by the addition of 5 μl Annexin V‐FITC and 10 μl propidium iodide. The mixture was incubated at room temperature in the dark for 30 min. After that, cell apoptosis was analyzed using flow cytometry.

At 24 h after seeding 5.0 × 10⁴ 293T cells in each well of a 24-well plate, CRISPR-Cas3 or -Cas9 expression plasmids were transfected as described above. Two days after transfection, the total cell number was counted using the Countess Automated Cell Counter (Thermo Fisher Scientific). Subsequently, apoptosis assays were performed with the Annexin V-FITC Apoptosis Detection Kit (Nacalai Tesque) according to the manufacturer’s protocol. FITC-positive cells were detected by FACS Aria IIIu (Becton Dickinson).