Tali apoptosis assay kit annexin 5 alexa fluor 488

The procedure was performed according to the manufacturer's protocol by using Tali™ apoptosis assay kit/Annexin V Alexa Fluor^® 488 and propidium iodide (PI) (Invitrogen/Life Technologies). After trypsinization, cells were centrifuged and resuspended in 100 μl Annexin binding buffer. Next, to each 100 μl of sample, 5 μl of Annexin V Alexa Fluor 488 was added. After 20 min of incubation in the dark, the cells were centrifuged, resuspended in Annexin binding buffer. Subsequently, 1 μl of PI to each sample was added and the cells were incubated at room temperature in the dark for 3 min. The data were analyzed using the FCs express Research Edition Software (ver4.03; De Novo Software, Los Angeles, CA, USA). The results revealed that the viable cells were Annexin V Alexa Fluor 488 and PI-negative; apoptotic cells were Annexin V Alexa Fluor 488-positive and PI-negative and Annexin V Alexa Fluor 488 and PI-positive; whereas necrotic cells were Annexin V Alexa Fluor 488-negative and PI-positive.