Propidium iodide

The CRC cells were incubated in 6-well plates with RC48 and/or GEM for 48 h. For cell apoptosis analysis, cell death was detected by using an Annexin V-FITC apoptosis kit (Beyotime Biotechnology, Shanghai, China). For cell cycle analysis, the cells were stained with propidium iodide (Beyotime Biotechnology, Shanghai, China), and total DNA content was detected with propidium iodide. Then cell cycle and apoptosis were measured by a Flow cytometer (BD, FACSCantoII, USA).

AGS and HGC-27 cells were transfected as described in section “Cell Transfection” and cultured for another 24 h to perform the apoptosis analysis. The cells were harvested and washed with ice-cold PBS for twice, and then the cell concentration was adjusted to 1 × 10⁶ cells/ml. Next, cells were stained with 5 μl Annexin V-FITC (Biyuntian, Shanghai, China) for 10 min. And, the cells were centrifuged at 1,000 × g for 5 min to remove the supernatant, and then suspended in binding buffer. Finally, the cells were stained with 10 μl propidium iodide (Biyuntian, Shanghai, China) for 5 min in the dark and analyzed by flow cytometry (BD Biosciences) to evaluate the rate of apoptosis.

Following the applied treatment, retinal cells were harvested, washed and resuspended in binding buffer with 1 μL of Annexin V-FITC and 1 μL of propidium iodide (PI) (Biyuntian, Wuxi, China). After incubation for 20 min at room temperature, cells were then tested by flow cytometry via CellQuest software (BD Biosciences, Shanghai, China). Annexin V ratio was recorded [8 (link), 39 (link)].

All reagents were of analytical grade and obtained from several different vendors. FeCl₃, NH₄SO₄, NH₄HPO₄, polyethylenimine (PEI, molecular weight = 25,000 Da), N-hydroxysuccinimide, and dialysis bag (3,500 Da) were purchased from Aladdin Biochemical Technology Co., Ltd (Shanghai, China), China. The hyaluronic acid (HA, molecular weight = 5,000–10,000 Da) was purchased from Zhanxun Biotechnology Co., Ltd (Xi’an, China). Dimethyl sulfoxide (DMSO) and absolute ethyl alcohol were purchased from Luoyang Chemical Regent Factory (Luoyang, China). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliun bromide (MTT), doxorubicin hydrochloride (DOX), and dulbecco minimum essential medium (DMEM) medium was purchased from Yuanye Bio-Technology Co., Ltd (Shanghai, China). The calcein acetoxymethyl ester (Calcein-AM) and Propidium Iodide (PI) were obtained from Biyuntian Biotechnology (Shanghai, China). The 4T1 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. Milli-Q water (18.2 MΩ·cm) was used in all experiments.