Click it plus edu proliferation kit

Cell proliferation was assessed using the Click-iT® Plus EdU Proliferation Kit (Thermo Fisher Scientific). Thirteen-day-old male mice were injected with 50 mg 5-ethynyl-2´-deoxyuridine (EdU) per kg bodyweight (5 mg/ml EdU solution in PBS) and sacrificed after 2 hours. Bones were fixed, decalcified and paraffin-embedded as described below. Paraffin sections were stained according to the manufacturer’s protocol.

Cell viability assay was performed using CCK-8 Kit (Dojindo). Briefly, the cells which were seeded into 96-well plates at a density of 1×10⁴ cells/well, were detected with 10 μL CCK-8, and incubated at 37℃ for 4h. The absorbance was measured at 450 nm. In this study, the proliferation index = the absorbance of experimental group—the absorbance of blank group, was used to evaluate the cell viability of each group. Besides, cell proliferation ability was evaluated with Click-iT® Plus EdU Proliferation Kit (Alexa Fluor® 555, Thermo Fisher Scientific), and the assay was mainly performed according to the manufacturer’s instructions.
To further evaluate cell viability, the extracellular/intracellular LDH, intracellular glycogen and glucose in medium in each group were measured with LDH-Cytotoxicity Colorimetric Assay KitⅡ(BioVision), Glycogen Colorimetric/Fluorometric Assay Kit (BioVision) and Amplex® Red Glucose Assay Kit (BioVision) according to the manufacturer’s instruction respectively.

According to the manufacturer’s instructions, 15 × 10³ cells were seeded into 6-well plates, and proliferation was analyzed 72 h after transfection using the Click-iT Plus EdU Proliferation Kit (ThermoFisher Scientific, Merelbeke, Belgium, #C10633). Briefly, the cells were incubated for 6 h with 5′-ethynyl-2′-deoxyuridine at 37 °C in the dark, and EdU incorporation was analyzed by flow cytometry on a BD LSRFortessa cell analyzer (FACS).