Click it edu cell proliferation assay kit

EdU Detection—EdU (5-Ethynyl-2’-deoxyuridine, EdU) is a thymidine analog that is incorporated into the DNA of dividing cells to indicate DNA synthesis [95 (link)]. The Click-iT EdU cell proliferation Assay Kit (Invitrogen, C10337) was used according to the manufacturer’s protocol and as previously described. Proliferation of enteroid crypts in larvae reared with and without RJ was measured using EdU to label cells in the S phase of the cell cycle. EdU reagent (10 μM) was added into the D-4 diet, and fed honey bee larvae for 24 h. EdU reagent (10 μM) was mixed well with honey to feed NWs and ELWs for 24 h. The midguts of larvae, NWs and ELWs were dissected in 1 × PBS, and fixed in 4% paraformaldehyde for 25 min at room temperature. After rinsing twice with 3% Bovine serum albumin (BSA) in PBS, the samples were incubated in 0.5% Triton X-100 in PBS for 20 min at room temperature, and then washed once with 3% BSA in PBS. These midguts were incubated with freshly arranged Click-iT reaction cocktail containing azide-conjugated Alexa Fluor 488 for 30 min at room temperature, protected from light. The samples were washed once with 3% BSA in PBS, and then incubated with 1 μg/ml DAPI for 30 min at room temperature in the dark. These midguts were mounted in mounting medium.

Cell proliferation was determined using the flow cytometry technology and Click-iT EdU Cell Proliferation Assay kit (Invitrogen, USA). Cells were seeded in 96-well plates at a density of 5000 cells/well and allowed to adhere for 24 h. The cells were then treated with NBQX, D-AP5, and LY341495 at various concentrations for 24 h. After treatment, 20 μM EdU was added to each well, and the cells were incubated for an additional 30 min at 37 °C. Cells were fixed with 3.7% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 for 20 min. EdU-labeled cells were visualized using Alexa Fluor 594 dye according to the manufacturer's instructions. The cells were counterstained with DAPI for 5 min at room temperature. The percentage of proliferating cells was calculated by dividing the number of EdU-positive cells by the total number of DAPI-stained cells in six randomly selected microscopic fields per well and identification and analysis of DAPI, EdU and Oct4 by high content screening. All experiments were repeated more than three times.

The cells were fixed using a fixation solution and subjected to senescence-associated β-galactosidase (SA-β-gal) staining following the manufacturer’s instructions (Biovision, Waltham, MA, USA). Imaging was performed using a Nikon Eclipse E800 microscope, and the number of positive cells was recorded. Cell proliferation was assessed by incorporating 5-ethynyl-2′-deoxyuridine (EdU) using the Click-iT EdU Cell Proliferation Assay Kit (Invitrogen). Quantification was performed using FIJI/ImageJ software (Version: 2.9.0/1.53t).

At 0, 12, 24, and 48 h after differentiation stimuli, cell proliferation was detected using incorporation of 5-ethynyl-2′-deoxyuridine (EdU) with the Click-iT EdU Cell Proliferation Assay Kit (Invitrogen). Briefly, cells were incubated with 10 μM EdU for 1 h before fixation, permeabilization, and EdU staining, which were carried out according to the kit’s protocol. The cells were incubated in a DAPI solution for 5 min. Fluorescence photographs were taken on EVOS^® FL Auto (Life Technologies; Carlsbad, CA, United States). Quantification of proliferation nuclei was accomplished by performing counts for DAPI and EdU. Nuclei were counted manually using digital photography and Adobe Photoshop software.

For cell proliferation assay, pregnant mice were intraperitoneally injected with 10 mM EdU (Invitrogen) in PBS (5 mg per 100 g body weight). Embryos were harvested 4 hours later and fixed in 4% paraformaldehyde for 30 min at 4°C, and were embedded in OCT compound. Cell proliferation was assessed on 6 µm frozen sections using Click-iT EdU Cell Proliferation Assay Kit (Invitrogen). To quantify cell proliferation, 8 ureter sections were prepared from each embryo, and sections are from comparable locations in the control and mutant. Two embryos of each genotype were analyzed. Proliferation index was calculated as the percentage of EdU positive cells relative to ureteral mesenchymal cells and ureteric epithelial cells, respectively. Apoptosis assay was performed on 6 µm frozen sections using in situ Cell Death Detection Kit (Roche) according to the manufacturer's instructions. Nine representative ureter sections from each of three embryos with control or mutant genotype at comparable locations were used for apoptosis assay. Apoptosis index was calculated as percentage of TUNEL positive cells relative to ureteral mesenchymal cells.

Cells were fixed and processed for senescence‐associated β‐galactosidase (SA‐β‐gal) staining as per the manufacturer's instruction (Biovision). Skin tissues, frozen in OCT, were cut (7–8 μm sections) and processed for SA‐β‐gal and Fast Red staining, as described (Velarde, Demaria, Melov, & Campisi, 2015; Wiley et al., 2016). A Nikon Eclipse E800 microscope was used for imaging and images were quantified using Image J software.
Cell proliferation was evaluated by incorporation of 5‐ethynyl‐2′‐deoxyuridine (EdU) and the Click‐iT EdU Cell Proliferation Assay Kit (Invitrogen). Briefly, cells were given 10 μM EdU for 24 hr before fixation, permeabilized and incubated with Click‐iT reaction cocktail as per the manufacturer's instructions. A Zeiss LSM780 confocal microscope was used for imaging, and images were quantified using Image J software. >100 cells from 5–7 different fields were quantified per condition, and all experiments were done in duplicate.