Bromodeoxyuridine brdu

To evaluate cell apoptosis in various types of cells, Her2-positive cell lines (BT474 and NCI-N87) were seeded at a density of 2 × 10⁶ cells/well in 12-well plates and exposed to Mil40, Mil40-E-15C, and MMAE at various concentrations (0.033, 0.1, 1 and 10 nM) for 24 h. Apoptosis and cell death were detected using an Annexin V-FITC Apoptosis Kit (Dojindo, Japan) and propidium iodide (PI) staining with FACSCalibur (BD Biosciences). For cell cycle position analysis after drug exposure, the cells were treated as described above and allowed to incorporate bromodeoxyuridine (BrdU; Beyotime Biotechnology, Shanghai, China) for 20 min. Nascent DNA synthesis was detected with anti-BrdUrd FITC, and total DNA content was detected with PI. Cell cycle position and apoptosis analyses were measured using FACSCalibur (BD Biosciences).
To detect the activity of Caspase3/7, Her2-positive cell lines (BT474 and NCI-N87) were seeded at a density of 6 × 10³ cells/well in 96-well plates and exposed to the drugs abovementioned. After incubation for 48 h, 100 μL Caspase3/7 reagents were added to each well. The plates were then placed on a horizontal shaker and incubated at room temperature for 1 h. The chemiluminescence values of each well were read using a TECAN spark 10M multifunctional enzyme marker.

For the colony formation assay, cells were seeded in specified numbers (100 cells/well) in 12-well plates to adhere overnight. Then the cells were co-treated with Nif (0–20 μM) and Pal (0–5 μM) for additional 7 days. The cells were fixed and stained with a 0.5% crystal violet solution for 15 min and the colonies were observed under a microscope. For bromodeoxyuridine (BrdU) analysis, cells in 96-well after indicated treatment for 3 days were pulsed with 10 µM BrdU (Beyotime, Shanghai, China) for 5 h, trypsinized, fixed, and labeled according to the manufacturer’s instructions.

To evaluate cell apoptosis, SKOV3 cell lines in 100 μL complete medium were plated in 12-well plates at a density of 5 × 10⁴ cells/well. Conjugate A4 at various concentrations (0, 100 and 300 nM) were added in each well and incubated under growth conditions for 48 h. To detected apoptosis and cell death, Annexin V-FITC Apoptosis Kit (Dojindo, Japan) and propidium iodide (PI) were used to stain, and the fluorescence intensity were measured by BD FACSAria II.
To evaluate cell cycle, SKOV3 cell lines in 100 μL complete medium were plated in 12-well plates at a density of 5 × 10⁴ cells/well. Conjugate A4 at various concentrations (0, 100 and 300 nM) were added in each well and incubated under growth conditions for 48 h. And bromodeoxyuridine (BrdU; Beyotime Biotechnology, Shanghai, China) was added and incubated for 20 min. To detected Nascent DNA synthesis and total DNA content, anti-BrdUrd FITC and phycoerythrin (PE) were used, respectively. To detected cell cycle position, FACSCalibur (BD Biosciences) was used. The above experimental methods refer to previously reported (Xiao et al., 2021 (link)).