Annexin 5 fluorescein isothiocyanate fitc propidium iodide apoptosis detection kit

Cell apoptosis was analyzed by an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Nanjing KeyGen Biotech, Co., Ltd., Nanjing, China) following the manufacturer's protocol. After transfection for 48 h, cells were harvested by 0.25% trypsin and centrifuged at 251 × g for 5 min at room temperature. Subsequently, cells were washed twice with cold PBS and resuspended in 250 µl binding buffer (Nanjing KeyGen Biotech, Co., Ltd.). FITC Annexin V FITC (5 µl) and 1 µl PI solution were added and cells were incubated for 15 min at room temperature. The apoptosis rate was detected using a Calibur Flow Cytometer (BD Biosciences, San Jose, CA, USA). Three independent experiments were repeated.

To further verify the effect of salidroside on the apoptosis of osteosarcoma cells, an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was used. Briefly, following treatment with (0, 1, 5 and 10 mM) salidroside for 48 h, MG63 cells (1×10⁵ cells/sample) were digested with trypsin (0.25%, room temperature, 1-3 min), centrifuged (300 × g, 4°C, 5 min), washed twice with PBS, and resuspended in binding buffer (500 μl/sample, Nanjing KeyGen Biotech Co., Ltd.). Each sample was stained with Annexin V-FITC (5 μl) and propidium iodide (PI; 5 μl) in the dark at 4°C for 15 min and then immediately analyzed with a flow cytometer equipped with FACSComp software (BD Biosciences, Franklin Lakes, NJ, USA). Annexin V-FITC^-/PI^- cells were identified as viable cells, Annexin V-FITC⁺/PI^- cells were identified as early apoptotic cells, and Annexin V-FITC⁺/PI⁺ cells were identified as the sum of late apoptotic cells and necrotic cells.

The Annexin V-fluorescein isothiocyanate (FITC)/Propidium Iodide (PI) Apoptosis Detection Kit (KeyGen Biotech, Nanjing, China) was utilized to examine cell apoptosis. In brief, A549 and H1299 cells were reacted with 500 µL binding buffer, followed by infiltrating with 5 µL Annexin V-FITC and 5 µL PI for 15 min in the dark. Eventually, the apoptotic cells were monitored using Attune NxT Flow Cytometer (Invitrogen).

Cell apoptosis was detected by an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's instructions. In brief, TIB-152 cells were seeded in 6-well plates (2×10⁵ cells/well) with RPMI-1640 supplemented with 10% FBS. Tan IIA (20 µM), IM (5 µM) or IM (5 µM) + Tan (20 µM) was added and the cells were cultured for 24 h. The TIB-152 group was treated with 0.1% DMSO. For rescue experiments, TBI-512 cells were pre-treated with IGF-1 (10 nM) 24 h before exposure to Tan IIA or/and IM. After 24 h of incubation, the cells were collected, centrifuged at 1,000 × g for 5 min and then resuspended in 500 µl binding buffer (provided with kit), followed by the addition of Annexin V-FITC (5 µl) and PI (5 µl). The samples were then incubated in the dark at room temperature for 15 min. Cell apoptosis assay was performed within 1 h on a flow cytometer (FACSCalibur system) equipped with Cell Quest software (version 5.1; BD Biosciences). The total percentage of apoptotic cells was defined as the sum of early and late apoptotic cells (22 (link)).

An Annexin-V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was used to detect the apoptosis of LX-2 cells, according to the manufacturer's protocol. LX-2 cells were seeded in 6-well plates at ~5×10⁴ cells/ml and subsequently serum-starved overnight when cells reached 30–50% confluence. LX-2 cells were only treated with AST (ranging between 5 and 80 µM) at 37°C for 12, 24, 48 and 72 h. However, LX-2 cells were transfected with miR-29b mimics or mimic negative control or inhibitors or inhibitor negative control at 37°C for 48 h and subsequently AST (40 µM) or the vehicle (DMSO) were added to the refreshed medium for 48 h. Following treatment, LX-2 cells were washed with cold PBS, collected by centrifugation (2,000 × g), and suspended in 500 µl 1× binding buffer and subsequently incubated with 5 µl Annexin V-FITC and 5 µl PI for 15 min at room temperature in the dark. Subsequently, apoptosis was analyzed with a FACS Calibur flow cytometer (BD FACSCalibur™; BD Biosciences, San Jose, CA, USA). A minimum of 10,000 cells per sample were acquired and analyzed using FlowJo software (version 7.6.1; Tree Star, Inc., Ashland, OR, USA). The experiments were repeated three times.