Apoalert annexin 5 fitc kit

5-Aminolevulinic acid (ALA) and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (St Louis, MO, USA). Necrostatin-1 (Nec-1), z-DEVD-fmk (ZDEVD) and z-IETD-fmk (ZIETD) were from Santa Cruz Biotechnology (CA). Cu-oxidised LDL (ox-LDL) and red fluorescent marked DiI-ox-LDL were both purchased from Peking Union-Biology Co. Ltd (Beijing, China). Hoechst-33342, DAPI, the ATP assay kit, the lactate dehydrogenase (LDH) release assay, and the caspase-3 and caspase-8 activity assay kit were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). The ApoAlert Annexin V-FITC kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Other drugs and chemicals used in this study were obtained from Sigma-Aldrich. EGS (ethylene glycol bis(succinimidyl succinate)) was obtained from Thermo Scientific.

Cell early stage apoptosis was detected by Annexin V-FITC assay [57] (link). The percentages of apoptosis of human or insect cells were determined by counting visible annexin V-positive cells under the fluorescence microscope. Cells (5,000 cells well⁻¹) were incubated with protein fractions of P. taiwanensis at 10 µg/ml or with P. taiwanensis (MOI = 1000) for 72 h on the well in 24-well plates. After treatment for 72 h, the cells were washed twice in PBS and detected using the ApoAlert Annexin V-FITC Kit (BD) according to the manufacturer's instructions. The DNA in the nuclei was stained with 4′,6-diamidino-2-phenylindole dilactate (DAPI) for 5 min. Finally, the stained cells were washed twice in PBS, fixed with 4% paraformaldehyde for 10 minutes, and then observed under a fluorescence microscope (Zeiss Axiovert 100M, Carl Zeiss, Germany). Annexin V positive cells were counted and identified as P. taiwanensis-induced early stage apoptotic cells.