Edu cell proliferation assay kit

The EDU Cell Proliferation Assay kit (Invitrogen, USA) was used to analyze cell multiplication. Following transfection 48 h, cells were seeded into 96-well plates at a concentration of 5,000 cells per well.and continue to cultivate for 24 h,after this, 2 h incubation with 50 µM EDU in 5% CO2 at 37 ° C and the cells are washed thrice with PBS, fixed using 4% polyacetaldehyde for 30 min, then incubated with 0.5% Triton-X-100 PBS for 20 min, finally stained with Apollo Dye Solution. Results were observed using high-content imaging microscopy.

Cells were seeded in 24‐well plates incubated with different concentrations of rhLRG‐1 (0, 300 ng/ml) under normoxic or hypoxia conditions. Twenty‐four hours after incubation, cell proliferation was detected using the EdU Cell Proliferation Assay Kit (Invitrogen, USA) according to the manufacturer's protocol. Briefly, cells were incubated with 50 mM EdU for 2 h before fixation, permeabilization and EdU staining. Then, cell nuclei were stained with DAPI (Sigma‐Aldrich, St. Louis, MO) at a concentration of 1 mg/ml for 8 min. The proportion of cells that incorporated EdU was determined by Zeiss 710 laser‐scanning microscope (Zeiss, Oberkochen, Germany).

Cell viability was determined using both MTS assay and EdU proliferation assay. NHEKs were seeded at a density of 5×10⁴ cells/well in a 96-well plate. After 24 hours, the media were replaced with fresh media containing 0μM, 1μM, 5μM, 10μM rottlerin. Concentrations of DMSO, which were used to dissolve the rottlerin, were maintained at <0.2% (v/v) among different treatments. In our experiment, a DMSO concentration of 0.2% didn’t affect keratinocyte proliferation, differentiation and death balance in the culture conditions. Cellular proliferation was measured after 24h of rottlerin exposure. When running the assay, reagents from a MTS reagent-based kit (Promega, USA) were added directly into the incubation media and incubated at 37°C for 1h. Absorbance at 490 nm was then measured using a plate reader (BioTek, USA). For the EdU proliferation assay, cells were seeded (n = 1×10⁴ cells per well) in 24-well plates. The cells were then incubated under standard conditions in complete media. Cell proliferation was detected using the incorporation of 5-ethynyl-29-deoxyuridine (EdU) with the EdU Cell Proliferation Assay Kit (Invitrogen, USA) according to the manufacturer’s protocol. The cell nuclei were stained with DAPI (Roche) at a concentration of 5ug/ml for 10 min. The proportion of cells that incorporated EdU was determined using by fluorescence microscopy (Leica, German).

Cell viability was determined using both MTS assay and EdU proliferation assay. NHEK were seeded (n = 2000 cells per well) in a 96-well microtiter plate and then infected with lentivirus as described above. Twenty-four hours after transfection, the transfected cells were grown in 100 μl complete medium. For MTS assay, briefly, proliferation was studied every 24 h up to a period of 5 d, at which point 100 μl of 0.5 mg/mL MTS solution (Promega) was added to each well and incubated for 3 h at 37°C. Formazan absorbance was read at 490 nm using a plate reader. For EdU proliferation assay, cells were seeded (n = 1× 10⁴ cells per well) in 24-well plates. Transfection of the cells was performed the following day as described above. 24 hours after transfection, the cells were incubated under standard conditions in complete media. Cell proliferation was detected using the incorporation of 5-ethynyl-29-deoxyuridine (EdU) with the EdU Cell Proliferation Assay Kit (Invitrogen). Briefly, the cells were incubated with 50 mM EdU for 3 h before fixation, permeabilization and EdU staining, which were performed according to the manufacturer’s protocol. The cell nuclei were stained with DAPI (Roche) at a concentration of 5ug/ml for 10 min. The proportion of cells that incorporated EdU was determined by fluorescence microscopy (Leica, German).