Annexin 5 fitc propidium iodide apoptosis detection kit

An Annexin V-FITC/Propidium Iodide (PI) Apoptosis Detection Kit (Dojindo Laboratories, Kumamoto, Japan) was used for the VSMC apoptosis assay. After a 3-day treatment period, VSMCs were removed from culture plates via trypsin digestion and washed twice with PBS. Following resuspension, the cells were double-stained with annexin V-FITC and PI and analysed via flow cytometry (FACSCanto, BD Biosciences, San Jose, CA, USA). After gating, the apoptosis rate was determined as the sum of the cell populations in quadrant 3 (Q3) (FITC+/PI−) and Q2 (FITC+/PI+). For Hoechst staining, VSMCs were treated for 3 days and subsequently stained with Hoechst 33258 (Merck, Kenilworth, NJ, USA) for 10 min in the dark. Morphological changes such as chromosomal condensation and nuclear fragmentation were counted in five different fields under a fluorescence microscope (magnification ×200).

flow cytometry assay was carried out to detect cell apoptosis. In brief, cells were transfected with si-NC+inhibitor-NC, si-LUCAT1+inhibitor-NC, si-LUCAT1+inhibitor-miR-181c-5p, and si-NC+inhibitor-miR-181c-5p. Following 48 hours of incubation at 37°C, the cells were harvested and subjected to apoptosis detection by using the Annexin V(FITC)/propidium iodide (PI) apoptosis detection kit (Dojindo, Japan). The cell apoptosis rate was determined by using the flow cytometry (Beckman Coulter, CA, USA) and analyzed using FlowJo 7.6 software (Tree Star, Inc., Ashland, OR, USA).

For apoptosis analysis, both apoptotic and necrotic cells in kidney differentiation cultures were measured using the Annexin V FITC/propidium iodide (PI) apoptosis detection kit (Dojindo) following the manufacturer’s protocol at 21–25 days. In brief, both adherent and floating cells were collected, 10⁶ cells were washed twice with cold sterile PBS and then resuspended in Annexin V-FITC binding buffer. FITC-conjugated Annexin V (100 μl/sample) was added and cells were incubated for 10 minutes at RT in the dark. Cells were then centrifuged and resuspended in binding buffer, and PI was added (100 μl/sample). Samples were kept on ice and incubated for 20 minutes in the dark. The total percentage of apoptotic cells was measured by counting the number of FITC⁺ and FITC⁺/PI⁺ stained cells by Guava easyCyte6HT™ flow cytometry (5000 events/gate). Representative data from one of three independent experiments were analyzed using its built-in INCYTE (version 2.7) software (EMD Millipore, Merck).

Seventy-two hours after cell transfection, Annexin V-FITC/Propidium Iodide (PI) Apoptosis Detection Kit (Dojindo, Japan) was used for apoptosis analysis in strict accordance with the manufacturer’s instructions. Then, the flow cytometer (BD Biosciences, USA) was used to differentiate the apoptotic cells. Annexin V (+)/PI(-) stained cells were considered as the viable apoptotic cells while Annexin V(+)/PI(+) stained cells represented the apoptotic cells. Apoptosis rate was calculated using the ratio of apoptotic cells / total cells × 100%.