Annexin 5 fluorescein isothiocyanate fitc 7 aminoactinomycin d 7 aad kit

GEM (crude drug) was obtained from Eli Lilly and Company (Indianapolis, IN, USA). GEM-C14 and GEM-HSA-NP were prepared as reported previously.21 (link) NBMPR was purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Normal saline (NS) was purchased from Shanghai Baxter Healthcare Co., Ltd. (Shanghai, China). Roswell Park Memorial Institute (RPMI)-1640 culture medium, fetal bovine serum and Dulbecco’s phosphate-buffered saline (DPBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cell-Counting Kit-8 (CCK-8) was purchased from Dojindo (Kyushu, Japan). Annexin V-fluorescein isothiocyanate (FITC)/7-aminoactinomycin D (7-AAD) kit and propidium iodide (PI) kit were purchased from BD Biosciences (San Jose, CA, USA). RNeasy Mini kit was purchased from Qiagen NV (Venlo, the Netherlands). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit was purchased from Hoffman-La Roche Ltd. (Basel, Switzerland).

Annexin V-fluorescein isothiocyanate (FITC)/7-amino-actinomycin D (7AAD) kit (BD Biosciences, NJ, USA) was used to track CT26 cell apoptosis. The CT26 cells were treated with Fe³⁺, TA-Fe³⁺, DOC, DOC@TA-Fe³⁺, and DOC@TA-Fe³⁺ + DFO. After incubation for 24 h, the cells were collected by adding free ethylenediaminetetraacetic acid tyrisin and incubated with the Annexin V-FITC/7AAD detection kit. Finally, the cell apoptosis ratio was analyzed via flow cytometry (ACEA Pharma, San Diego, CA, USA).
The CT26 cells were plated in plates to further visualize the live and dead cells after treatment with Fe³⁺, TA-Fe³⁺, DOC, DOC@TA-Fe³⁺, and DOC@TA-Fe³⁺ + DFO. After treatment for 24 h, the cells were coincubated with calcein-AM and PI for 30 min in the dark to observe with CLSM.