Click ittm plus edu alexa fluor 647 flow cytometry assay kit

The detailed protocol is described in [46 (link)]. In brief, 92.1 cells (2.5 × 104) were plated on a 1.5% agar bed (w/v) for 5 days. Three-dimensional spheroids were transferred and embedded into collagen I solution. The next day, collagen-embedded 3D tumor spheroids were treated as described in figure legends. For cell viability, cells were incubated with calcein-AM (7 µM) and propidium iodide (PI, 10 μg/mL) for 30 min at the end of the experiment. Pictures were obtained using a Nikon A1R confocal microscope. For EdU incorporation, cells were incubated with analogue EdU (10 µM) for 16 h. Tumor spheroids were extracted from collagen solution using collagenase type 1 (Sigma-Aldrich) and EdU incorporation was measured using the Click-iT^TM Plus EdU Alexa Fluor^TM 647 flow cytometry assay kit (Thermo Fisher Scientific), following company instructions.

The EdU assay was performed using an EdU Kit (RiboBio, C10310-1). Irradiated IEC-6 cells were cultured with or without TT-2 (10 μg/mL) for 48 h in DMEM supplemented with 10% FBS, and then switched to fresh DMEM supplemented with EdU (50 μM) and incubated for 2 h; this was followed by fixation, permeabilization, and EdU staining with Apollo567 (RiboBio, C00031). The nuclei were stained with DAPI, and the staining of EdU-positive cells was observed using fluorescent reverse microscopy (UltraVIEW VOX, PerkinElmer). The percentage of incorporation of EdU in the nucleus of irradiated IEC-6 cells in each field was calculated and expressed as the mean ± SD. For flow cytometry assays, the cells were exposed to 10 μM EdU for 2 h at 37°C, and were prepared and treated using a Click-iT^TM Plus EdU Alexa Fluor^TM 647 Flow Cytometry Assay Kit (C10635, Thermo Fisher Scientific) according to the manufacturer’s instructions. Flow cytometry analysis was performed using the FACSCalibur platform (BD Biosciences) to detect EdU incorporation.