Edu labeling detection kit

According to instructions from the EdU Labeling/Detection Kit (Ribo-bio, Guangzhou, China), SCs (2.5 × 10⁴/cm²) were plated onto 24-well plates and incubated in medium containing 10 μM EdU for 48 hours at 37°C under 5% CO2. Subsequently, the SCs were fixed with 4% paraformaldehyde for 30 minutes. After rinsing with PBS, SCs were incubated with 200 μL of 1× Apollo^® reaction cocktail per well at 37°C for 30 minutes, permeated with 0.2% Triton X-100 in PBS, and stained with Hoechst 33342 dye (5 g/mL) for 30 minutes. Images were observed under a fluorescence microscope (Olympus Imaging Systems). The percentage of EdU-positive cells was calculated using Image Pro Plus 5.0 software (Media Cybernetics, Silver Spring, MD, USA) in five random fields of three samples.

According to the EdU Labeling/Detection Kit manual (Ribobio, Guangzhou, China), Schwann cells were cultured in 24-well plates at 5 × 10⁴ cells per well and incubated in 10 μM EdU labeling medium for 48 hours at 37°C in a humidified 5% CO₂ incubator. At the appropriate time, cultured cells were fixed with 4% paraformaldehyde for 30 minutes. After three PBS washes, staining was performed with 200 μL of 1 × Apollo^® reaction cocktail at 37°C for 30 minutes. Following permeabilization with 0.2% Triton X-100 in PBS, the cells were stained with 5 g/mL Hoechst 33342 dye for 30 minutes, and observed under a fluorescence microscope (Olympus). The percentage of EdU-positive cells was calculated.

The proliferative ability was performed using CCK-8 and EdU assay. For the CCK-8 assay, HLECs were seeded in 96-well culture plates and administrated with 10 μL of CCK-8 assay kit (Dojindo Japan). 24 h later, cells were measured for the absorbance at 450 nm. For the EdU assay, HLECs were seeded in 24-well plates at 2 × 10⁴ cells using the EdU labeling/detection kit (Ribobio, Guangzhou, China). EdU labeling medium was added and fixed in 4% paraformaldehyde (pH 7.4) for 30 min. After counterstaining with 250 mL DAPI (Invitrogen, Molecular Probes, Eugene, OR, USA) for 25 min, EdU-positive cells were imaged under a fluorescence microscope (Nikon Corporation, Tokyo, Japan).

According to the manual of a EdU labeling/detection kit (RiboBio, Guangzhou, People’s Republic of China), 50 μM EdU labeling medium was added to the cell culture to allow incubation for 2 hours at 37°C under 5% CO₂. Afterwards, the cells were fixed with 4% paraformaldehyde (pH 7.4) for 30 minutes and incubated with glycine for 5 minutes. After washing with phosphate-buffered saline (PBS), staining with anti-EdU working solution was performed at room temperature for 30 minutes. Following wash with 0.5% Triton X-100 in PBS, the cells were incubated with Hoechst33342 (5 μg/mL) at room temperature for 30 minutes, followed by observation under fluorescent microscopy. The percentage of EdU-positive cells was calculated from five random fields in three wells.