Beyoclick edu cell proliferation kit with alexa fluor 647

Proliferation of cells was detected with a BeyoClickEdU Cell Proliferation Kit with Alexa Fluor 647 (Beyotime, Shanghai, China) following the manufacturer’s protocol. Briefly, cells were seeded into 12-well plates at a density of 10000/well and incubated with siRNA or vehicle. After exposure to nomorxia or hypoxia for 48h, cells were treated with EdU (20 µm) for 3 h and subjected to fixing and permeabilization. Then, cells were exposed in click additive solution for 30 min followed by Hoechst staining for 10min, and finally observed with fluorescence microscope (Olympus, Japan).

For immunofluorescence staining, cultured TSPCs were fixed in 4% paraformaldehyde for 15 min at room temperature. Cells were blocked with 10% normal serum blocking solution (3% bovine serum albumin and 0.1% Triton X-100 and 0.05% Tween-20) for 2 h at room temperature. After being washed, cells were incubated overnight at 4 °C with anti-AQP1 (Proteintech) and p16^INK4A (Abcam), followed by a mixture of Alexa Fluor 594-conjugated secondary antibodies (Molecular Probes) was incubated 2 h at room temperature. Immunofluorescence was visualized with a Nikon Ts2R fluorescence microscope (×20 or ×40 objectives). For EdU detection, the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 647 was used according to the manufacturer’s protocol (Beyotime Biotechnology). Immunofluorescence was visualized with an Olympus FV1000 confocal microscope (×40 objectives).

LX2 cells were treated with the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 647 (Cat#: C0081S, Beyotime, Shanghai, China) for the determination of proliferation according to the instruction for use. Hoechst 33342 (5 μg/mL, Cat#: C0031, Solarbio) was employed to re-stain the cell nucleus. The pictures were photographed by a fluorescence microscopy (Olympus, Tokyo, Japan).

Cell proliferation assay was performed using the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 647 (Beyotime). Briefly, the cells were seeded in 96-well plates at a density of 5 × 10³ cells/well for 48 h after transfection and then treated with indicated drugs. Then, the cells were incubated with 10 μM EdU for 2 h at 37°C. After being fixed with 4% paraformaldehyde for 30 min, the cells were treated with 0.1% Triton X-100 for 10 min and rinsed with PBS three times. Thereafter, the cells were exposed to 100 μl of click reaction cocktail for 30 min and then incubated with 5 μg/ml of Hoechst 33342 to stain the cell nuclei for 30 min. Images were captured using Olympus IX73 microscope. The percentage of EdU-positive cells was defined as the proliferation rate. All the experiments were performed in triplicate.

KYSE30 or KYSE510 cells in six-well plates were trypsinized, washed once with PBS, and fixed in cold 70% ethanol at 4 °C overnight before being stored for up to one week. The cells were then washed once with PBS and incubated with propidium iodide (PI) (50 μg/mL) and RNase A (100 μg/mL) in PBS in the dark for 30 min at 37 °C. For cell cycle analysis, the cells were treated with 10 μM EdU for 30 min, trypsinized, and washed once with PBS before being fixed in cold 70% ethanol at 4 °C overnight and stored for up to one week. The samples were then analyzed using a BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 647 (C0081L, Beyotime Biotechnology, Shanghai, China). Briefly, the cells were washed once in PBS with 2% BSA and incubated in PBS containing 2% BSA, PI (50 μg/mL), and RNase A (100 μg/mL) in the dark for 30 min at 37 °C. The cells were then selected using a flow cytometer (BD Biosciences) with side scatter (SSC) and forward scatter (FSC). DNA staining with PI reflected the cell cycle G1, S, and G2/M stages. DNA staining with EdU reflected the S-phase of the cell cycle.

5-ethynyl-2′-deoxyuridine (EdU) uptake assay was also used to determine the cell proliferation. In briefly, cells transfected with shDHX9 or shControl were incubated with EdU (10 μM) for 2 hours in complete medium. After treatment with a fixing solution and a permeabilizing solution, the cells were stained with anti-EdU antibody and Propidium Iodide (PI). EdU staining was performed with BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 647 (Beyotime, CN), according to the manufacturer’s protocol. Stained cells were run on a FACS Calibur (Beckman, US). The data were analyzed using FlowJo 10.6.2 software.

6 × 10⁵ cells in each well were plated into 6-well plates, and the cells were cultured at 37 °C with 5% CO₂. To assess the capacity for cell proliferation, the cells were stained using a BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 647 (C0081S, Beyotime, Shanghai, China). Hoechst 33,342 (5 µg/mL, C0031, Solarbio) was used to identify cell nucleus. Fluorescence microscopy (Olympus, Tokyo, Japan) was used to capture the images.