Cell light edu apollo567 in vitro kit

Mouse primary keratinocytes were isolated from newborn fetuses, as described previously 23 (link). The cells were then incubated with primary K15 or K19 antibody. Next, the cells were incubated with secondary antibodies labeled with Alexa Fluor 546 and Hoechst 33342, as described previously 24 (link).
Mouse primary keratinocytes were isolated from Cdc42^{loxp/loxp-Cre-} neonates. The cells were then infected with viruses (vector, plenti-CMV-NLS-Cre (plenti-Cre), 1 × 10⁹ infectious units/ml, purchased from OBiO Technology (Shanghai) Corp., Ltd.); after 3 days, the expression of Cdc42 was detected by Western blotting (WB). Then, the primary keratinocytes were cultured in high-calcium medium (Ca²⁺ concentration increased from 0.09 to 2 mM) for cell experiments (cell-cell junctions and SPRR family members) as described previously 25 (link).
The proliferation of primary keratinocytes was detected with a 5-ethyl-2′-deoxyuridine (EdU) assay (Cell-Light EdU Apollo 567 In Vitro Kit, Ribobio, China). Apoptotic primary keratinocytes were detected using a TUNEL BrightRed Apoptosis Detection Kit (Vazyme Biotech Co., Ltd., China).

Mice were injected with EdU (5 mg/kg) intraperitoneally 6 h before sacrifice. The colon tissues were embedded in paraffin for the subsequent staining. Cell proliferation was assessed by the Cell-Light EdU Apollo 567 In Vitro Kit (RiboBio, Guangzhou, Guangdong, China. Cat. #C00003), according to the manufacturer’s instructions. In brief, the tissue was permeabilized with 0.5% Triton X-100 and reacted with 1 × Apollo reaction cocktail for 30 min. Subsequently, the DNA contents of the cells were stained with DAPI for 30 min and visualized under a fluorescence microscope (Leica Microsystems GmbH, DMI6000B).

A Cell-Light Edu Apollo 567 In Vitro Kit (RiboBio, Guangzhou, China) was used to detect the amount of cells present in the DNA synthesis phase. 3T3-L1 preadipocytes were subcultivated in 96-well plates at a density of 4000 cells per well and treated with EdU Reagent A (RiboBio, Guangzhou, China) for 4 h. Hoechst stain was used for cell nuclei staining. Images were captured and analyzed with a Nikon TE2000 microscope (Nikon, Tokyo, Japan).

5-ethynyl-2′ deoxyuridine (EdU) assay was performed using a Cell-Light EdU Apollo567 In Vitro Kit (RiboBio, Guangzhou, China). Briefly, GCs were seeded in 96-well plates at 2 × 10³ per well. GCs were treated with dexamethasone at 100 nmol/L for 2 h. Then, GCs were treated with overexpression plasmids or siRNA for 24 h and incubated with 50 μmol EdU for 2 h. GCs were washed twice with PBS, fixed with 4% paraformaldehyde for 30 min, neutralized with 2 mg/mL glycine for 5 min, and then permeabilized with 0.5% TritonX-100 for 5 min. GCs were incubated in a mixture of reagents B, C, D, and E for 30 min. The cells were washed three times with 0.5% TritonX-100, followed by two washes with methanol. The nuclei were stained with Hoechst for 30 min. Finally, the cells were observed using a Nikon TE 2000 microscope (Nikon, Tokyo, Japan).

EdU incorporation assay was performed with the Cell-Light EdU Apollo567 In Vitro Kit (Ribobio, China) according the manufacturer’s instructions. Imaging was performed on an Olympus FV-1000 confocal microscope. Red nuclei EdU cells were examined by randomly counting 10 fields in the middle of the microscope slide and were expressed as a percentage of the total population. Three independent assays were carried out.

We used the F4 generation of WT and PFN2a-overexpressing cells for EDU assays. WT and PFN2a-overexpressing cells were seeded in the 48-well plates at 2 × 10⁴/cm², respectively. After 24 h of incubation in growth medium (GM), these C2C12 cells were used for Edu labeling by Cell-Light EdU Apollo567 In Vitro Kit (C10310-1, RiboBio, Guangzhou, China) according to the manufacturer’s instructions. The EdU-stained cells were visualized by using a Nikon TE2000-U inverted microscope (Nikon Instruments, Tokyo, Japan). Myoblast proliferation (ratio of EdU⁺ to all myoblasts) was counted using Nikon Instruments.

Cell proliferation assay is based on incorporating 5-ethynyl-2′-deoxyuridine (EdU) into genomic DNA, which was analyzed using Cell-Light EdU Apollo567 In Vitro Kit (Riobio, Guangzhou, China). Apollo staining and Hoechst 33342 staining (for nuclear staining) were performed. Fluorescence images were analyzed using ImageJ (NIH, Bethesda, MD, United States). The EdU incorporation rate was equal to the ratio of EdU-positive cells (red)/total number of Hoechst-positive cells (blue).