EdU Cell Proliferation kit (Cat#C10339, Invitrogen) was used to determine the cell proliferation. After starvation, HUVECs were cultured with EdU-labeling mixture (10 mM) under hypoxia for 12 h. Then cell proliferation was detected according to the manufacturer’s instruction. In summary, 4% paraformaldehyde was used to fix HUVECs, and 0.5% TritonX‐100 was used to permeabilized them. Subsequently, HUVECs were incubated with 1 × Click-iT EdU reaction mixture and stained with Hoechst 33342 for another 15 min. The cell proliferation rate was calculated as EdU-positive cells/total cells of each field.
Edu cell proliferation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EdU Cell Proliferation Kit is a laboratory tool used to detect and quantify cell proliferation. It utilizes 5-ethynyl-2'-deoxyuridine (EdU), a thymidine analog, which is incorporated into the DNA of dividing cells during the S phase of the cell cycle. The kit provides a simple, efficient, and sensitive method for visualizing and analyzing cell proliferation in various experimental systems.
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Lab products found in correlation
Alexa fluor 596 imaging kit, by Thermo Fisher Scientific (2 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Gapdh 14c10, by Cell Signaling Technology (1 mentions)
Snap surface 488, by New England Biolabs (1 mentions)
Hoescht, by Merck Group (1 mentions)
Non phospho active β catenin, by Cell Signaling Technology (1 mentions)
Lamin b1 antibody 12987 1 ap, by Proteintech (1 mentions)
Texas red egf, by Thermo Fisher Scientific (1 mentions)
E cadherin antibody hecd 1, by Takara Bio (1 mentions)
Notch1 ecd abs90, by Merck Group (1 mentions)
Fam83h antibody, by Fortis Life Sciences (1 mentions)
Alexa fluor 647 azide, by Thermo Fisher Scientific (1 mentions)
β catenin antibody, by BD (1 mentions)
Ab32575, by Abcam (1 mentions)
One step tunel apoptosis assay kit, by Beyotime (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Beyotime (1 mentions)
Automated microscope, by Agilent Technologies (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Incucyte, by Sartorius (1 mentions)
Wnt c59, by Selleck Chemicals (1 mentions)
13 protocols using edu cell proliferation kit
1
Quantifying Cell Proliferation with EdU
2
EdU Cell Proliferation Assay for GSCs
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The assay was performed according to instructions from EdU Cell Proliferation Kit (C10337, Invitrogen, USA). Briefly, indicated GSC1 were seeded at a density of 6×104 per well into 48-well plates. After 24 h culture, 50 μM EdU solution was diluted in the cell culture medium, followed by adding into indicated GSC cells and then incubated at 37° C for another 2 h. The BrdU positive ratio determined by imageJ. Cell proliferation assay was performed as previously before [21 (link)]. The significance of difference was determined by the last day of the experiments.
3
Quantifying bMSC Proliferation with EdU
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Cell proliferation was evaluated using EdU Cell Proliferation Kit (Invitrogen; AF488 for Imaging), as described.35 Briefly, bMSCs were cultured in medium with AGE‐BSA or BSA (400 μg/mL) with 5% O2 at 37 ℃ for 24 hours, and then moved to the medium with 1× 5‐ethynyl‐2'‐deoxyuridine (EdU) solution and incubated for 6 hours. After washing (×3), cells were fixed and permeabilized, and then mixed with Click‐iT reaction cocktail for imaging. Proliferative cells were examined using a fluorescence microscope and quantified using software ImageJ.
4
Notch1, EGFR, and β-Catenin Signaling Assays
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Antibodies against Notch1 ICD (D1E11, 1:100 IF, 1:1,000 WB), Notch1 V1744 (D3B8, 1:500 WB), EGFR (D38B1, 1:100 IF, 1:1,000 WB), pEGFR (D7A5, 1:1,000 WB), GFP (D5.1, 1:1,000 WB), non-phospho (active) β-catenin (D13A1, 1:1,000 WB), GAPDH (14C10, 1:10,000 WB), and YAP (D8H1X, 1:200 IF) were from Cell Signaling Technologies. β-catenin antibody (14, 1:1,000 WB) was from BD Biosciences. E-cadherin antibody (HECD-1, 1:1,000 IF, 1:1,000 WB) was from Takara Bio. Notch1 ECD (ABS90, 1:1,000) was from Millipore. FAM83H antibody (1:1,000 WB) was from Bethyl Laboratories. Lamin B1 antibody (12987-1-AP, 1:1,000) was from Proteintech. TexasRed-EGF, rhodamine phalloidin, and Alexa Fluor 488, 568, and 647 goat anti-mouse and anti-rabbit IgG secondary antibodies (1:400) were from Invitrogen. Alexa Fluor 647 azide and EdU cell proliferation kit were from Invitrogen. Hoescht and DAPT were from Sigma. Anti-SNAP (P9310S, 1:1,000), SNAP-Capture, and SNAP-Surface 488 were from New England Biolabs.
5
Therapeutic Potential of EVs in Cardiac Repair
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MI/R mice were randomized into three groups who received intravenous injection of PBS, EVs (100 μg EVs per mouse), or P-EVs (containing 100 μg EVs per mouse) after reperfusion every 7 days for up to 4 weeks. At treatment day 7, EdU (50 mg/kg, Beyotime) was injected via the tail vein before the heart was harvested to label proliferating cells. Proliferation was measured using EdU Cell Proliferation Kit (Invitrogen, C10339). The heart sections were also stained using One Step TUNEL Apoptosis Assay Kit (C1090, Beyotime) to detect cell apoptosis. All cryostat sections were double-stained with anti-CD31 antibody to count CD31/EdU or CD31/TUNEL double-positive cells. qRT-PCR was performed to detect the expression of angiogenesis-related genes in the hearts of mice at treatment day 7. The primers used in this assay are listed in Table S2 . At treatment day 28, the hearts were harvested and cut into 6 μm cryosections. Angiogenesis was evaluated by staining sections with rat monoclonal anti-CD31 antibody and rabbit monoclonal anti-α-smooth muscle actin (α-SMA) antibody (ab32575, Abcam). Cardiac cells were identified by staining sections with rabbit polyclonal anti-cTnT antibody.
6
Measuring Retinal Cell Proliferation in Mice
7
EdU Cell Proliferation Assay
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Cell proliferation was analyzed using the EdU Cell Proliferation Kit (Invitrogen), which detects only cells in the S-phase, according to the manufacturer’s instructions. Briefly, EdU labeling solution (1:500 dilution) was incubated at 37 °C for ≤ 8 h. Cells were imaged using a fluorescence microscope.
8
Quantifying Endothelial Cell Proliferation
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EPCs were cultured on coverslips in a 6-well plate containing 10 μM EdU for 5 h. EdU staining was conducted using the EdU Cell Proliferation Kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. The nuclei were stained with Hoechst-33342 (Sigma-Aldrich). An automated microscope (BioTek, Winooski, VT, USA) was used to acquire the images.
9
Evaluating Cell Proliferation Assays
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To evaluate cell proliferation, three distinct assays were employed: the colony formation assay, the 5‐ethynyl‐2′‐deoxyuridine (EdU) Cell Proliferation Kit (Invitrogen), and the Cell Counting Kit‐8 (CCK‐8, APExBIO) assay. For conducting the CCK‐8 assay, 4000 cells per well were seeded onto 96‐well plates and thereafter cultivated for a period of 24 h. Subsequently, 10 μL of CCK‐8 reagent was added to each well at 24, 48, 72, and 96 h, and the plate was left to incubate for 2 h in a 37°C incubator. Then, the absorbance at 450 nm was quantified through a microplate reader. For the EdU assay, NSCLC cells were seeded into 6‐well culture plates and analyzed based on the kit instructions. For the colony formation assay, cells in different groups were taken for digestion, blown repeatedly to form a single cell, and prepared into a cell suspension. Then, 200 cells per well were added to a 6‐well plate and placed in an incubator. After culturing for 2 weeks, formaldehyde was added for fixation and then stained with 0.1% crystal violet.
10
Cell Proliferation Analysis of hRECs with Exosomes
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Cell proliferation was measured using the cck-8 kit (MCE, USA) and the EdU Cell Proliferation kit with an Alexa Fluor 596 Imaging kit (Thermo Fisher Scientific) following the manufacturer's instructions. In brief, normal hRECs or hRECs at an initial density of 5 × 103 cells/well, cocultured with 100 μg/mL MH-exo or PDR-exo, were seeded into 96-well plates and transfected with various oligonucleotides for 48 h. The cell proliferation was evaluated by 450 nm absorbance values at 6, 12, 24, 48, and 72 h thereafter using an enzyme-linked immunosorbent assay plate reader. Data are presented as mean ± SD of five replicates. Regarding the EdU assay, 100 μL 50 μM EdU medium was added into each well and incubated for 2 h in 37°C. The cells were then washed twice using PBS for 10 min, fixed in 4% PFA for 15 min, neutralized with 2 mg/mL glycine, and washed with PBS before permeabilizing with 0.5% Triton X-100 for 10 min. Finally, the hRECs were labeled using 100 μL Apollo-596 staining agent and washed in 0.5% Triton X-100 three times. EdU assay was performed three times independently and a total of three randomly selected fields were imaged in each condition using a confocal fluorescence microscope (MIC00223 LSM5 Live). The percentage of EdU-positive cells (labeled red) was calculated and analyzed using ImageJ software.
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