Cell proliferation was assessed through the CCK-8 assay and EdU assay. For the CCK-8 assay, after 0, 24, 48, 72, and 96 h of cell transfection, hRMECs in 96-well plates were harvested after 2 h of incubation with 10 μL CCK-8 reagent (Dojindo Laboratories, Kumamoto, Japan). Cell viability was monitored by measuring OD450 using a microplate reader (BioTek). For the EdU assay, transfected hRMECs in 96-well plates were prepared for 3 h of incubation using the Cell-Light EdU Apollo488 In Vitro Kit (RiboBio, Guangzhou, China). EdU labeling medium was applied at a concentration of 50 μM EdU. After they were fixed and rinsed, cells were treated with 1 × Apollo 488 and Hoechst solution (5 μg/mL, 100 μL) in sequence for visualization under a fluorescence microscope (Olympus, Tokyo, Japan).
Cell light edu apollo488 in vitro kit
Manufactured by RiboBio
Sourced in China
The Cell-Light EdU Apollo488 In Vitro Kit is a laboratory product designed for detecting and visualizing DNA synthesis in cultured cells. It utilizes the EdU (5-ethynyl-2'-deoxyuridine) labeling method to incorporate a fluorescent dye into newly synthesized DNA. This kit provides the necessary reagents and protocols to perform this analysis.
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Lab products found in correlation
Lsm 510, by Zeiss (4 mentions)
Fluorescence microscope, by Olympus (3 mentions)
Fluorescence microscope, by Nikon (3 mentions)
Cck 8 solution, by Dojindo Laboratories (3 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (2 mentions)
Gapdh, by Proteintech (2 mentions)
Fsx100 microscope, by Olympus (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (2 mentions)
Fluorescence microscope, by Leica (2 mentions)
Microplate reader, by Agilent Technologies (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Cck 8 reagent, by Dojindo Laboratories (1 mentions)
Confocal microscope, by Olympus (1 mentions)
Sorafenib, by Selleck Chemicals (1 mentions)
Sorafenib, by Merck Group (1 mentions)
Lonafarnib, by Merck Group (1 mentions)
Chloroquine, by Selleck Chemicals (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
68 protocols using cell light edu apollo488 in vitro kit
1
Assessing Human Retinal Cell Proliferation
2
Quantifying Cell Proliferation with EdU Assay
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To determine the effect of CNPase on cell proliferation, a Cell‐Light EdU Apollo 488 In Vitro Kit (RiboBio, No.: C10337) was used according to the manufacturer's protocol. In brief, SRA 01/04 cells were seeded into 96‐well plates. Forty‐eight hours after transfection, reagent A was diluted with complete medium at a ratio of 1000:1; the cells were then labelled with EdU (100 μL/well) for 2 hours. The cells were washed with PBS and fixed with 50 μL/well of fixative (PBS containing 4% paraformaldehyde) at room temperature for 30 minutes. Next, the cells were incubated with 2 mg/mL glycine (50 μL/well) for 5 minutes. After washing with PBS (100 μL/well) for 5 minutes, the cells were incubated with 100 μL/well of penetrant (PBS containing 0.5% Triton X‐100) for another 10 minutes and then washed once with PBS (5 minutes). Subsequently, the cells were incubated with 1× Apollo staining solution (100 μL/well) and 1× Hoechst 33342 reaction solution in the dark for 30 minutes. After an additional 10 minutes of treatment with the penetrant, the cells were washed twice with methanol. Signals were detected using a confocal microscope (Olympus). Images of positively stained cells were captured and counted by ImageJ.
3
Investigating Autophagy Regulation via Targeted Compounds
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Lonafarnib (S2797), sorafenib (S7397), chloroquine (CQ, S4157), MG-132 (S2619) were obtained from Selleck Chemicals (TX, USA). Stock solution of 10 mM Lonafarnib, 20 mM sorafenib, 100 mM chloroquine and 100 mM MG-132 were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) and stored at −80°C. Cell Counting Kit-8 (CCK8) was purchased from Dojindo Molecular Technologies Inc. (Kumamoto, Japan). The mRFP-GFP-LC3B adenovirus construct was purchased from Hanbio Inc. (Shanghai, China). Cell-Light Edu Apollo488 In Vitro Kit was purchased from RiboBio Company (Guangzhou, China). Antibodies used were as follow: P62 (#88588, Cell Signaling Technology), ATG3 (#3415, Cell Signaling Technology), ATG7 (#8558, Cell Signaling Technology), cyclin D1 (#2978, Cell Signaling Technology), CDK4 (#12790, Cell Signaling Technology), CDK6 (#13331, Cell Signaling Technology), phosphor-Ser780-Rb (#8180, Cell Signaling Technology), LC3B (GTX127375, GeneTex), GAPDH (60004-1-1g, Proteintech).
4
CCK-8 Assay and Cell Viability Evaluation
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CCK8 assay: Cells were plated in 96-well plates (2000 cells/well in 100 µL), cultured overnight, and incubated with 0, 0.99, 2.96, 8.89, 26.67 and 80 μg mL−1 NPs for 24 h. Then, 10 µL of cell counting kit-8 (CCK-8, Dojindo, Japan) was added, and the cells were incubated at 37 °C in the dark for 4 h. The optical density at 450 nm was determined with a microplate reader. Live/dead, EdU, and ROS measurements: OCM1a cells were pretreated for 24 h with PBS, 15 µg mL−1 ICG-MOF-PR, ICG-OP@MOF-PR, ICG-Cis@MOF-PR and ICG-COP@MOF-PR. Cell survival (calcein AM staining) and death (PI staining) were assessed using a live/dead kit (Beyotime, China) according to recommended procedures. Cell proliferation was studied using a Cell-Light EdU Apollo488 In Vitro Kit (RiboBio, China) following the manufacturer’s instructions. The ROS levels were assessed using a DCFH-DA assay (Beyotime, China) according to recommended procedures. Images were taken with a fluorescence microscope (Nikon).
5
EdU-Based Cell Proliferation Assay
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EdU was detected using a Cell-Light Edu Apollo488 In Vitro Kit (RiboBio, C10310-3) according to the manufacturers’ protocol. Briefly, cells were seeded onto coverslips in 24-well plates at a density of 10,000 cells/well. Twenty-four hours later, the cells were incubated with EdU solution (50 μM) for 2 h and fixed with 4% paraformaldehyde for 30 min. After neutralization with 2 mg/ml glycine solution and washing with PBS buffer, cells were incubated with penetrant (0.5% Triton X-100 in PBS buffer) on a shaker for 10 min. Apollo solution was added and incubated for 30 min in the dark at room temperature. After washing twice with penetrant and methanol, the cells were stained with Hoechst 33342. Slides were sealed with an antifluorescence quenching reagent, and images were captured using a fluorescence microscope (Nikon).
6
Cell Proliferation Assay Using EdU
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Cells were seeded into a 24-well plate overnight, followed by incubation with Cell-Light EdU Apollo488 in Vitro Kit (RIBOBIO, #C10310-3, China) for 2 h. 4% formaldehyde was used to fix the cell after washing with PBS twice. Subsequently, glycine (2 mg/ml) (Sangon Biotech, #56-40-6, Shanghai, China) was added for 5 min, and 0.5% Triton-X-100(Sangon Biotech, #9002-93-1, Shanghai, China) was added into cells for 10 min incubation. Then, cells were treated with Apollo for 30 min and washed with 0.5% Triton-X-100 three times. Next, the cells were stained with Hoechst 33342 and protected from light for 30 min at room temperature. Cell proliferation was assessed by Fluorescence Microplate Reader (BioTek).
7
Quantifying Cellular Proliferation with EdU Assay
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A549 and H1975 SNORA38B-OE/KO cells were seeded on sterile cover glasses placed in the 24-well plates overnight, and incubated with 50 µM 5-ethynyl-2’-deoxyuridine (EdU) (C10310-3, Cell-Light EdU Apollo488 In Vitro Kit, RiBoBio) in the culture medium for 2 hours. After washing with 1×PBS, cells were then fixed for 20 min in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in 1×PBS for 10 min. Then, cells were incubated with 1×Apollo reaction buffer for 30 min at dark, followed by three times of washing using 0.5% Triton X-100 in 1×PBS, and another three times of washing by 1×PBS. Next, mounted the coverslip onto the slides with Antifade Mounting Medium with DAPI (P0131, Beyotime Biotechnology) and visualized using fluorescence microscope (Olympus 600 auto-biochemical analyzer). Ten microscopic fields were taken for calculating EdU, and images were analyzed using with Image-Pro Plus software.
8
Measuring Cell Proliferation by EdU Staining
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As a kind of thymidine analog, EdU incorporates into genome DNA in the S phase during DNA synthesis. The quantity of the incorporated EdU reflects cell proliferation activity. EdU staining of HCC cells was performed using the Cell-Light EdU Apollo488 In Vitro Flow Cytometry Kit or Cell-Light EdU Apollo488 In Vitro Kit (Ribobio). For the flow cytometry analysis, HCC cells were planted into 6-well plate at 3 × 105 (HepG2) or 1.5 × 105 (QGY-7703) per well. The cells were transfected, stained and analyzed using an Accuri C6 Plus flow cytometer (BD, San Jose, CA, USA). For imaging, the HCC cells were planted and treated similarly as the CCK-8 assay. The cells in 96-well plate were stained and captured under an IX71 fluorescence microscope (Olympus). Fluorescence intensity was quantified using ImageJ software. The EdU cytometry and imaging tests were performed in two independent experiments with each group in duplicate.
9
Quantifying Apoptosis and Proliferation in GSCs
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Apoptotic cell death was detected according to the protocol that was included with the TUNEL kit (Fluorescein In Situ Apoptosis Detection Kit, KeyGEN BioTECH, China). The GSCs grown on the polylysine-coated coverslips were treated with GO, and then the coverslips were fixed with 4% paraformaldehyde for 30 min. The coverslips were used for TUNEL staining. After labeling, images were acquired by fluorescence microscopy.
The EdU labeling assays were performed using the Cell-Light EdU Apollo 488 In Vitro kit (RiboBio, Guangzhou, China). GSCs were treated with 50 μM EdU for 2 h at 37 °C according to the manufacturer’s protocol. Then cells were fixed with 4% paraformaldehyde, permeabilized with 0.4% Triton X-100, and incubated with Apollo® reagent. The nuclear DNA was labeled with DAPI and representative images were obtained. The number of EdU- or DAPI-positive cells was counted with ImageJ.
The EdU labeling assays were performed using the Cell-Light EdU Apollo 488 In Vitro kit (RiboBio, Guangzhou, China). GSCs were treated with 50 μM EdU for 2 h at 37 °C according to the manufacturer’s protocol. Then cells were fixed with 4% paraformaldehyde, permeabilized with 0.4% Triton X-100, and incubated with Apollo® reagent. The nuclear DNA was labeled with DAPI and representative images were obtained. The number of EdU- or DAPI-positive cells was counted with ImageJ.
10
EdU Assay for Cell Proliferation
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The cells were cultured until reaching 50% density and transfected with different plasmids. The cells were subjected to the 5-ethynyl-2′-deoxyuridine (EdU) assay 20 h after transfection, using the Cell-Light EdU Apollo488 In Vitro Kit (RiboBio, Guangzhou, China). We captured three randomly selected fields using a fluorescence microscope (TCS SP8, Leica) and determined the number of EdU-stained cells. ImageJ software was utilized to determine the percentage of EdU-positive cells.
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