The cellular immunofluorescence staining for Lamin B1 (1:100, CL594-66095, Proteintech, China) was performed to detect cell senescence. For tissue immunofluorescence staining, after blocking, the frozen sections were incubated with primary antibodies against NeuN (1:1000, ab177487, Abcam, USA), Aβ (mouse anti-Aβ (1:200, SIG-39320, Covance), DCX (1:100, ab18723, Abcam, USA), MAB1281 (1:100, MAB1281, Merck, USA), GFAP (1:500, 16825-1-AP, Proteintech, China) and Iba1 (1:100, 10904-1-AP, Proteintech, China) overnight at 4 °C, and then incubated with Cy3 or FITC-conjugated anti-mouse or ant-rabbit IgG (1:200, AB0122, Abways, China) for 2 h. For EdU/DCX, EdU/NeuN, or EdU/Nestin double immunofluorescence staining, EdU solution (5 mg/kg) was intraperitoneally injected daily for three consecutive days before the mice were sacrificed, according to the instructions of the Cell-Light EdU Apollo567Kit (RiboBio, China). After Apollo staining, the slices were respectively incubated with DCX, NeuN or Nestin antibody (1:1000, 19483-1-AP, Proteintech, USA) overnight at 4 °C and incubated with FITC-conjugated IgG antibody (1:500, SA00003-2, Proteintech, USA) for 2 h. After DAPI (1:100) counterstaining, the sections were examined under a microscope (Leica, German), and the positive cells were analyzed using Image J software (Bethesda, USA).
Cell light edu apollo 567 kit
Manufactured by RiboBio
Sourced in China
The Cell-Light EdU Apollo 567 Kit is a laboratory equipment used for the detection and visualization of DNA synthesis in proliferating cells. It utilizes the incorporation of the thymidine analog EdU (5-ethynyl-2'-deoxyuridine) to label replicating DNA, which can then be detected through a copper-catalyzed click reaction with the Apollo 567 fluorescent dye.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Olympus (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Ab18723, by Abcam (1 mentions)
Ab177487, by Abcam (1 mentions)
Mab1281, by Merck Group (1 mentions)
Sa00003 2, by Proteintech (1 mentions)
Imagej software, by Leica camera (1 mentions)
Sig 39320, by Fortrea (1 mentions)
Cck8 kit reagent, by Beyotime (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (1 mentions)
A microplate reader, by Thermo Fisher Scientific (1 mentions)
Triton x 100 solution, by Merck Group (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Biosharp (1 mentions)
Inverted fluorescence microscope, by Olympus (1 mentions)
Dapi reagent, by Wuhan Servicebio Technology (1 mentions)
Fluorescence photomicroscope, by Zeiss (1 mentions)
16 protocols using cell light edu apollo 567 kit
1
Cellular and Tissue Immunofluorescence Staining
2
EdU Proliferation Assay Protocol
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For the EdU assay, 4 × 103 cells were seeded in a 96-well plate, and the Cell-Light EdU Apollo 567 Kit (RiboBio, China) was used to evaluate cell proliferation. After removing the medium, the cells were treated with a 50 μM EdU solution at 37°C. Then, the cells were fixed with 4% paraformaldehyde for 30 min and incubated with glycol for 5 min. After incubation with 100 μL 0.5% Triton X-100, the cells were washed with PBS, incubated with 100 μL Apollo staining reaction, and finally fixed and decolorized with methanol. Cells were visualized under a fluorescence microscope.
3
Evaluating Cell Proliferation and Apoptosis
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Cell proliferation was determined by Cell Counting Kit-8 (CCK8) and 5-ethynyl-2-deoxyuridine (EdU) assays. For the CCK8 assay, GES-1 and MKN28 cells were seeded in different conditioned medias at a density of 2000 cells per well in 96-well plates. Every 24 h for 72 h, 10 μL of the CCK8 kit reagent (C0038, Beyotime, China) was added to each well, and cell viability was measured with spectrophotometry at OD450nm after 2 h of incubation. The EdU assay was performed using the Cell-Light™ EdU Apollo® 567 kit (C10310-1, Ribobio, China). The cells were seeded into 24-well culture plates and treated with different medias or drugs, after which they were incubated with EdU for 2 h. Apollo and Hoechst staining solutions were used to observe EdU-positive cells according to the manufacturer’s instructions. Apoptosis was evaluated by FITC Annexin V Apoptosis Detection Kit (C1062L, Beyotime, China). The cells were stained with FITC-Annexin V and propidium iodide (PI) according to the manufacturer’s protocol. The apoptosis rate was detected by fluorescence-activated cell sorting (FACS) after 15 min of staining.
4
Cell Proliferation Assays: MTT and EdU
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For the MTT assay, 2 × 103 cells were seeded in a 96-well plate and cultured in complete medium. Ten microliters of MTT solution (5 mg/mL) was added to each well at the indicated time points. After incubation for 4 h, 100 μL of dimethyl sulfoxide (DMSO) was added to each well to stop the reaction. Thirty minutes later, the absorbance was measured using a microplate reader (Thermo) at 490 nm.
For the EdU assay, 4 × 103 cells were seeded in a 96-well plate, and the Cell-Light EdU Apollo 567 Kit (Ribobio, Shanghai, China) was used to evaluate cell proliferation. After removing the medium, the cells were treated with 100 μL of a 50 μM EdU solution at 37 °C for 2 h. Then, the cells were fixed with 4% paraformaldehyde for 30 min and incubated with 50 μL glycocoll for 5 min. After incubation with 100 μL of 0.5% Triton X-100 penetrant, the cells were washed with PBS and incubated with 100 μL of Apollo staining reaction at room temperature and then decolorized with methanol. The cells were visualized under a fluorescence microscope.
For the EdU assay, 4 × 103 cells were seeded in a 96-well plate, and the Cell-Light EdU Apollo 567 Kit (Ribobio, Shanghai, China) was used to evaluate cell proliferation. After removing the medium, the cells were treated with 100 μL of a 50 μM EdU solution at 37 °C for 2 h. Then, the cells were fixed with 4% paraformaldehyde for 30 min and incubated with 50 μL glycocoll for 5 min. After incubation with 100 μL of 0.5% Triton X-100 penetrant, the cells were washed with PBS and incubated with 100 μL of Apollo staining reaction at room temperature and then decolorized with methanol. The cells were visualized under a fluorescence microscope.
5
Quantifying Proliferating Transfected Cells
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Transfected cells (after 36 h) were immobilized with 4% paraformaldehyde (SHRBIO) and permeabilized with triton X‐100 solution (0.5%, Sigma‐Aldrich). Afterwards, cell nuclei were colored in accordance with the instructions of the Cell‐Light EdU Apollo 567 kit (RiboBio), while three random fields of view were used for image taking and counting under a fluorescent microscope (Olympus).
6
Cell Growth and Proliferation Assays
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For the cell growth curve, 1 × 104 cells were seeded to 6-well plates and cultured for 6 days. The cells were counted every day to draw the cell growth curve. The 5-ethynyl-2’-deoxyuridine (EdU) assay was performed with the Cell-Light EdU Apollo 567 kit (RiboBio, China) according to the manufacturer’s instructions. All images were photographed with a fluorescence microscope (Olympus, DP72). EdU quantification was performed as the ratio of the number of red fluorescence cells, i.e., EdU + , to the total number of DAPI + cells in a given field. At least six randomly selected fields from each sample were scored. All experiments were independently performed three times, totally 18 fields to analyze. For colony formation, cells were harvested and seeded into 6-well plates with amount of 1 × 103 cells/well. On day 10, the cell colonies were stained with Giemsa stain for 15 min after fixation with 4% paraformaldehyde (Biosharp, China) for 30 min. Colonies with > 50 cells were counted. Three independent experiments were performed.
7
Cell Proliferation Assay in 96-well Plates
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Cells in 96-well plates at a density of 5000 cells/well were analyzed utilizing a Cell-Light EdU Apollo 567 kit (RiboBio) according to the manufacturer’s instructions. We used an inverted fluorescence microscope (Olympus, Tokyo, Japan) to count cells in five random fields, with experiments performed in triplicate using independent samples.
8
Quantifying EdU-positive HASMCs
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HASMCs were treated according to the manufacturer's protocol (Cell-Light EdU Apollo567 Kit; Guangzhou RiboBio Co., Ltd.). EdU-positive cells were observed using an Axio Observer Z1 fluorescence microscope (Zeiss GmbH) and analyzed using ImageJ version 1.8.0 (National Institutes of Health).
9
Cell Proliferation Assay in H9C2 Cells
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H9C2 cells were seeded in 96-well plates at a density of 5x103 cells/well for 48 h and cell proliferation was evaluated using the Cell-Light EdU Apollo567 kit (Guangzhou RiboBio Co., Ltd.). Briefly, 50 µmol/l of EdU was added into the cell culture medium and incubated for 2 h. The cells were then treated with 4% paraformaldehyde for 30 min and 0.5% Triton X-100 for 20 min. After washing with PBS, the cells were stained at room temperature for 30 min with the anti-EdU working solution and incubated with 100 µl of the Hoechst 33342 stain (5 µg/ml). Finally, a fluorescence microscope (Olympus Corporation) was used to capture images of the cells, and the percentage of EdU-positive cells was calculated from 5 optical fields/well.
10
Evaluating Cell Proliferation with EdU Detection
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We used the Cell-Light EdU Apollo567 kit (C10310, Ribobio, China) to evaluate cell proliferation. According to the manufacturer's protocol, the cells were seeded in 3.5-cm-diameter dishes. When Cells were grown to 50 % confluency and starved with 1 % FBS medium overnight. Then, replaced the complete culture medium (10 % FBS) and treated with TGF-β1 and Wnt3a. After LIPUS treatment, a 50 μM EdU culture medium was prepared by diluting EdU solution 1000:1; each 3.5-cm-diameter dish was supplemented with 1 mL for 2 h, and then fixed with 4 % PFA for 20 min. After washing with PBS three times, the cells were stained with APOLLO. Next, the samples were incubated with DAPI for 15 min at 37 °C. Finally, three randomly selected fields were captured using a fluorescence microscope (Olympus, Japan). In vivo, mice were intraperitoneally injected with 5 mg/kg EdU solution 4 h before harvest. Then, the joint tissue was decalcified in 15 % EDTA for 2 weeks. After that, all samples were embedded in frozen section medium and sectioned with a freezing microtome. The staining and imaging methods were the same as those used for the in vitro study.
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