Cell counting kit 8 cck 8 assay kit

Cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8) assay kit (AR1191, Boster Biological Technology., LTD, Wuhan, China) and colony formation assays. For the CCK-8 assay, cells (1000 cells/well) were plated in 96-well plates and treated with CCK-8 solution at 0 h, 24 h, 48 h, and 72 h. The absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay reader (DNM9606, Beijing Perlong Technology Co., Ltd, Beijing, China).
In the colony formation assay, cells were treated with 0.25% trypsin-EDTA (T1300, Solarbio Co., Ltd. Beijing, China). The reaction was stopped by adding complete medium containing 10% FBS (tb-534237681231, GIBCO, New York, USA). After 1–2 weeks of incubation, cells were fixed with 4% paraformaldehyde for 30 min at 4 °C and stained with 0.5 mL of 0.1% crystal violet (C8470, Solarbio Co., Ltd. Beijing, China). Subsequently, cells were rinsed with PBS, and colonies were photographed.

Cells (3 × 10³ cells/well) were seeded in 96‐well plates overnight. Anlotinib or gefitinib was added at different doses, and the cells were incubated for 72 hours. Then, a Cell Counting Kit‐8 (CCK‐8) assay kit (Boster, Wuhan, China) was used according to the manufacturer's instructions to assess cell viability. Each sample was plated in triplicate, three independent experiments were performed, and IC50 was defined as the concentration needed for a 50% reduction in the absorbance.

Dendrobium officinale was purchased from the herbal medicine market in Chongqing, China and was authenticated by Professor Xiaojun Sun from Beijing Huadafangke Commodity Quality Inspection Co. Ltd. (Beijing, China) before the preparation of AuNPs. Adriamycin (ADM) was obtained from Shenzhen Main Luck Pharmaceuticals Inc. (Shenzhen, China). Chloroauric acid (HAuCl₄) was purchased from Shanghai Xianding Biological Technology Co. Ltd. (Shanghai, China). Trisodium citrate dihydrate was gained from Beijing G-CLONE Biological Technology Co. Ltd. (Beijing, China). The cell counting kit-8 (CCK-8) assay kit was purchased from Boster Biological Technology Co. Ltd. (Pleasanton, CA). Dulbecco's modified Eagle's medium (DMEM) and 0.25% EDTA/trypsin were procured from HyClone (Logan, UT). Fetal bovine serum (FBS) was purchased from Gibco Life Technologies (Carlsbad, CA).