Annexin 5 fitc kit

Cell apoptosis was analyzed with Annexin V-FITC kit (Nanjing Keygen Biotech. Co., Ltd). Briefly, FITC-conjugated Annexin V (50 μl/well) and propidium iodide (PI, 50 μl/well) were added to the cells infected by AMP-treated viruses. Then, the obtained mixture was incubated at room temperature for 15 min in the dark prior to fluorescence observation. Cell apoptosis was determined on basis of the observation soon after initiating apoptosis. Cells translocated the membrane phosphatidylserine (PS) from the inner surface of the plasma membrane to the cell surface. Thereafter, PS can be easily detected by staining with a fluorescent conjugate of Annexin V (green), a protein that has a high affinity to PS. Cellular late apoptosis was determined by cell staining with PI (red). The results were analyzed by fluorescence microscope (Nikon, Japan) at 36 hpi.

Apoptosis was detected using the Annexin V-FITC kit (KeyGen Biotech; CAT No. KGA108) as described previously [23 (link)]. Briefly, BMDMs were treated with vehicle or CB-839 (1 nM to 1 μM) for 24 h, harvested, washed, and incubated with Annexin V-FITC and propidium iodide for 15 min at room temperature in the dark. Cells were immediately analyzed using a BD FACSCalibur flow cytometry system (BD Biosciences, San Jose, CA, USA).

MCF-7 cells were obtained from the American Type Culture Collection (ATCC). Lipofectamine 2000 Reagent was purchased from Invitrogen (Carlsbad, California, USA). 5-FU and DAPI were purchased from Sigma (St. Louis, MO, USA). Anti-Bcl-2, anti-Bax, anti-Caspase 3, anti-GAPDH were obtained from Santa Cruz. Anti-ERK and anti-p-ERK, anti-JNK and anti-p-JNK were obtained from Cell Signaling (Boston, MA, USA). Cell Counting Kit-8 (CCK8) was purchased from Bytotime Company (Nantong, Jiangsu Province, China). Annexin V/FITC kit was purchased from KeyGEN BioTECH (Nanjing, Jiangsu Province, China). Chemiluminesence (ECL) assay kit was purchased from Amersham (Arlington Heights, USA).