Annexin 5 fluorescein isothiocyanate fitc apoptosis staining detection kit

Apoptosis was measured by flow cytometry using the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Staining/Detection Kit (Abcam, Inc.) according to the manufacturer’s protocol. In brief, the collected cells were double-stained with annexin V and propidium iodide (PI) in the dark, and annexin V-positive cells were regarded as apoptosis-induced cells using a flow cytometer (BD Biosciences, San Jose, CA, USA). MMP was evaluated with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) dye following general manufacturer’s recommendations. The fluorescent intensities for JC-1 monomers and aggregates forms were analyzed by flow cytometry, and then percentages of JC-1 monomers were expressed to represent cells that lost MMP.

CRISPR/Cas9 cell types (3×10⁵ viable cells/well) were seeded into 6‐well plates in MCDB131 containing 10% FBS. After 72 hours, the culture medium was removed, and the cells were washed with PBS. Accutase (STEMCELL Technologies, Cambridge, MA; cat. no. 07920) was used to detach the cells. The cells were washed and centrifuged at 300g for 5 minutes and resuspended at 1×10⁶ cells/mL in 500 µL of 1× binding buffer (annexin V‐fluorescein isothiocyanate (FITC) Apoptosis Staining/Detection Kit; Abcam, Cambridge, UK; cat. no. ab14085). The cells were transferred to 12×75‐mm tubes and annexin V‐FITC/propidium iodide was added and incubated for 5 minutes at 22°C, protected from light. Stained cell suspensions were analyzed by flow cytometry using a BD LSRFortessa X20 (BD Biosciences).