We assessed fibroblast proliferation using a Cell‐Light 5‐ethynyl‐2‐deoxyuridine (EdU) Apollo567 In Vitro Kit (Ribobio Co., Ltd.) according to the manufacturer's instructions. Briefly, fibroblasts (2 × 104/well) were cultured in a Millicell EZ SLIDE 8‐Well glass slide (Merck Millipore Ltd.) and then incubated with 50 μmol/L EdU (1:1000) for 12 hour. Fibroblasts were fixed with 4% formaldehyde for 20 minutes at 37°C, followed by permeabilisation in 0.5% Triton X‐100 at 37°C. Then, 100 μL of Apollo® reaction cocktail was added to each well and incubated for 30 minutes under light‐shading conditions. After three washes with PBS, the nuclei were counterstained with 4′,6′‐diamidino‐2‐phenylindole (DAPI) for 20 minutes at 37°C, and the EdU‐labelled cells were observed by a laser scanning confocal microscopy (Leica SP8) and normalised to the total number of DAPI‐stained cells.
Apollo567 in vitro kit
Manufactured by RiboBio
Sourced in China
The Apollo567 In Vitro Kit is a laboratory equipment product designed for in vitro applications. It provides the core functionality required for such experiments.
Automatically generated - may contain errors
Lab products found in correlation
Cell light 5, by RiboBio (4 mentions)
Millicell ez slide 8 well glass slide, by Merck Group (2 mentions)
Ix73 aizfl ph, by Olympus (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Annexin 5 pe 7 aad apoptosis detection kit, by BD (1 mentions)
Tunel assay, by Beyotime (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Hoechst, by Merck Group (1 mentions)
Anti gfap antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Abcam (1 mentions)
Inverted fluorescence microscope, by Leica (1 mentions)
11 protocols using apollo567 in vitro kit
1
Fibroblast Proliferation Assay using EdU
2
Quantifying Cell Proliferation with EdU
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Cell growth was detected by Cell-Light 5-ethynyl-2′-deoxyuridine (EdU) Apollo 567 In vitro kit (RiboBio, Guangzhou, China). After transfection, the cells were cultured in a 96-well plate (1 × 104/well) for 48 h, followed by incubation with an EdU-labeled medium of 50 μM for 2 h. Then, the cells were immobilized in 4% paraformaldehyde, permeated in 0.5% TritonX-100/PBS, and stained using Apollo solution and Hoechst33342. An inverted fluorescence microscope IX73-AIZFL/PH (OLYMPUS Corporation, Japan) was used to generate cell images.
3
Comprehensive Analysis of Cell Proliferation and Apoptosis
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Cell proliferation was analyzed using a Cell-Light 5-ethynyl-2′-deoxyuridine (EdU) Apollo567 In Vitro Kit (RiboBio, Shanghai, China) in strict accordance with the manufacturer's instructions. Images were acquired at 20 × magnification with a fluorescence microscope (Leica, Solms, Germany). EdU-positive cells were counted in the selected cells and calculated as the mean value from multiple fields of view. Cell apoptosis assays were performed using an Annexin V-PE 7-AAD Apoptosis Detection Kit (BD Biosciences, CA, USA) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL assay, Beyotime, Shanghai, China) according to the manufacturer's protocol. For Annexin V-PE 7-AAD Apoptosis assay, the cells were analyzed the percentage of apoptotic cells, that cells undergoing early and late apoptosis, among 10,000–20,000 counted cells using a flow cytometer (BD Biosciences, CA, USA). For TUNEL assay, images of apoptosis cells were obtained using a microscope (Leica, Solms, Germany).
4
Astrocyte Proliferation Assay with EdU
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Astrocyte proliferation was determined using the Cell-Light EdU (5-ethynyl-2′-deoxyuridine) Apollo567 In Vitro Kit (#C10310; Riobio, Guangzhou, China) following the manufacturer’s instructions. SB216763 or Nec-1 were added to the medium during reoxygenation and the astrocytes were incubated for 24 h. Cells were sequentially incubated with 20 μM EdU during reoxygenation; 4% paraformaldehyde (to fix the cells for morphological analysis) for 30 min at room temperature; and 0.5% Triton X-100 (to permeabilize the cells) for 10 min. Finally, 1× Apollo reaction solution was added and the cells were incubated for 30 min. To label GFAP, cells were exposed overnight to an anti-GFAP antibody (1:1,000, #53-9892-82, Thermo, MA, USA) at 4°C. Hoechst (1:5,000; #33258; Sigma-Aldrich, MO, USA) was used to stain the nuclei. Images were obtained using a confocal microscope (LSM 710; Carl Zeiss, Jena, Germany).
5
Measuring FLS Proliferation with EdU Assay
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We assessed FLS proliferation using a Cell-Light 5-ethynyl2-deoxyuridine (EdU) Apollo567 In Vitro Kit (Ribobio Co., Ltd). According to the manufacturer's instructions. Briefly, FLSs (2 × 104/well) were cultured in a Millicell EZ SLIDE 8-Well glass slide (Merck Millipore Ltd.) and then incubated with 50 μmol/L EdU (1:1000) for 24 h. FLSs were fixed with 4 % formaldehyde for 30 min at 37 °C, followed by permeabilization in 0.5 % Triton X-100 at 37 °C. Then, 100 μL of Apollo® reaction cocktail was added to each well and incubated for 30 min under light-shading conditions. After three washes with PBS, the nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) for 30 min at 37 °C, and the EdU-labeled cells were observed by laser scanning confocal microscopy (Leica SP8).
6
Proliferation Quantification of N9 Cells
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The proliferation of the N9 cells was determined using a Cell-Light EdU (5-ethynyl-20-deoxyuridine) Apollo 567 In Vitro Kit (RiboBio, China) according to the product instructions. The percentage of EdU-positive cells was calculated from five random fields in three wells.
7
EdU-based Cell Proliferation Assay
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Cell proliferation was analysed using Cell‐Light 5‐ethynyl‐2’‐deoxyuridine (EdU) Apollo 567 in vitro kit (Guangzhou RiboBio Co., Ltd.). The transfected cells were cultured in a 96‐well plate (1 × 104 cells/well). After 48 hours of culture, 50 μmol L−1 of EdU‐labelled medium was added, followed by incubation for an additional 2 hours. The cells were then fixed in 4% paraformaldehyde (PFA), permeabilized in 0.5% Triton×‐100/PBS, and stained with Apollo staining solution and Hoechst33342. Cell images were obtained under an inverted fluorescence microscope IX73‐AIZFL/PH (Olympus Optical Co., Ltd.).
8
Cell Proliferation Assay with EdU
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The CMs were incubated with 5-ethynyl-2'-deoxyuridine by using Apollo 567 in vitro Kit (Ribobio, China). Briefly, cells were incubated with 5-ethynyl-2'-deoxyuridine for 12 h and fixed with 4% paraformaldehyde at 37 °C for 15 min. Next, the Apollo mixture reagent was added to evaluate DNA synthesis, then counterstained with DAPI.
9
Quantifying Cell Proliferation via EdU
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To detect cell proliferation ability, EdU Apollo567 In Vitro kit (RiboBio, C10310-1) was used. After treatment, HUVECs were incubated with 50 μM EdU for 2 h. Cells were fixed using 4% paraformaldehyde (PFA) and stained with dye solution according to manufacturer's protocol. DAPI (Thermo Fisher Scientific, 62248) was used for labeling cell nuclei.
10
Cell Proliferation Measurement via EdU Assay
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At 24 hours after transduction, cell proliferation was measured using a Cell-Light 5-ethynyl-2?-deoxyuridine (EdU) Apollo567 In Vitro Kit (Ribobio) according to the manufacturer's protocol, as previously described (Zheng et al., 2020). Briefly, the cells were exposed to EdU for 2 hours, then washed and fixed with 4% paraformaldehyde for 30 minutes. Following washing with phosphate-buffered saline, the cells were blocked with phosphate-buffered saline containing 0.5% Triton X-100 for 10 minutes. Cells were incubated with 200 μL of 1× Apollo® staining reaction solution for 30 minutes, and the nuclei were stained with Hoechst 33342 (Abcam, Cambridge, MA, USA, Cat# ab228551, 1:1000).
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