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Edu imaging kit

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The EdU imaging kit is a product designed for the detection and visualization of DNA synthesis in cells. It utilizes the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into newly synthesized DNA, which can then be detected using a fluorescent dye. This kit provides the necessary reagents and protocols for performing this analysis.

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Lab products found in correlation

5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (5 mentions) Dmi3000b microscope, by Leica camera (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (4 mentions) Cck 8, by Beyotime (4 mentions) Multiscan fc, by Thermo Fisher Scientific (3 mentions) 4 6 diamidino 2 phenylindole (dapi), by Promega (2 mentions) Leica sp5, by Leica camera (2 mentions) Deadend fluorometric tunel system, by Promega (2 mentions) Ix71 inverted fluorescence microscope, by Olympus (2 mentions) Microplate reader, by Thermo Fisher Scientific (2 mentions) Lab tech chamber slide, by Thermo Fisher Scientific (2 mentions) Crystal violet, by Beyotime (1 mentions) Cck 8 kit, by Dojindo Laboratories (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Mda mb 231 human breast cancer cells, by American Type Culture Collection (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Z1 coulter particle counter, by Beckman Coulter (1 mentions) Imager m2 microscope, by Zeiss (1 mentions) Fluorescent microscope, by Olympus (1 mentions) Ab34771, by Abcam (1 mentions)

25 protocols using edu imaging kit

1

Quantifying Cell Proliferation and Colony Formation

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For 5-ethynyl-2-deoxyuridine (EdU) assay, 24-well culture plates were applied to culture transfected cells. The newly synthesized DNA was visualized with an EdU imaging kit (Life Technologies) to assess cell proliferation. The number of EdU positive cells was calculated under a fluorescence microscope.
For colony formation assay, transfected cells were resuspended at 1×103 cells/well and cultured in culture for about 4 days until macroscopic colonies appeared. Finally, the colonies were stained with crystal violet (Beyotime) for 30 mins.
Qi H., Xiao Z, & Wang Y. (2019). Long non-coding RNA LINC00665 gastric cancer tumorigenesis by regulation miR-149-3p/RNF2 axis. OncoTargets and therapy, 12, 6981-6990.
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2

Evaluating Proliferative Ability of GBM Cells

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CCK-8 and plate colony formation assays were performed to evaluate the proliferative ability of the cells22 (link),24 (link). The cells were seeded in 24-well plates overnight and then transfected with miR-NC, miR-129-5p or miR-129-5p plus pcDNA3.1-Wnt5a. GBM stem cells or monolayer cells were trypsinized and seeded in 96-well plates at a confluence of 2000 cells per well per 100 μL of stem cells or 10% FBS supplemented DMEM. Absorption of the cells was measured at different indicated time points using the CCK8 kit (Dojindo Laboratories, Kumamoto, Japan) following the manufacturer’s instructions. An EdU imaging kit (Life Technologies) was used to determine DNA synthesis of cells grown on coverslips in a 24-well dish after appropriate TMZ treatments. Immunostaining and EdU assay results were visualised using a Leica DMI3000B microscope. EdU-positive cells were manually counted.
Zeng A., Yin J., Li Y., Li R., Wang Z., Zhou X., Jin X., Shen F., Yan W, & You Y. (2018). miR-129-5p targets Wnt5a to block PKC/ERK/NF-κB and JNK pathways in glioblastoma. Cell Death & Disease, 9(3), 394.
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3

Quantifying Cell Proliferation and Apoptosis

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Cell proliferation and cell apoptosis were determined using EdU and TUNEL assays, respectively, as described previously28 (link). For cell proliferation assay, pregnant female mice were injected with EdU (Life Technology) through IP at a concentration of 100 mg/kg. After a 2 h pulse, the hearts were collected and processed for frozen sections as described above in the Immunostaining Section. The serial sections crossing the whole heart were first stained with IB4 or PECAM1 and ELASTIN antibody followed by EdU staining with EdU imaging Kit (Life Technology) and counterstained with DAPI (Vector lab). The stained sections were photographed using a Zeiss Observer Z1 or Leica SP5 confocal microscope. EdU-positive cells were counted using Image J and the data was presented as the ratio of EdU-positive cells among total cells. Three hearts were analyzed for each genotype. Apoptotic cells in E14.5 hearts of embryos were visualized by TUNEL assay. The frozen sections of isolated hearts were prepared as described in the EdU assay. Serial sections were first stained with IB4 or PECAM1 and ELASTIN antibody, followed by TUNEL assay using DeadEnd™ Fluorometric TUNEL System (Promega) and counterstained with DAPI. The stained sections were photographed using a Zeiss Observer Z1 or Leica SP5 confocal microscope.
Lu P., Wang P., Wu B., Wang Y., Liu Y., Cheng W., Feng X., Yuan X., Atteya M.M., Ferro H., Sugi Y., Rydquist G., Esmaily M., Butcher J.T., Chang C.P., Lenz J., Zheng D, & Zhou B. (2022). A SOX17-PDGFB signaling axis regulates aortic root development. Nature Communications, 13, 4065.
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4

Radioluminescence and Fluorescence Imaging of MDA-MB-231 Cells

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MDA-MB-231 human breast cancer cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in DMEM (Gibco) medium supplemented with 10% fetal bovine serum. Glass-bottom dishes were coated with fibronectin (10 µg/ml) for 1h. The dish was then washed 3× with PBS, and 105 cells were seeded and left to adhere overnight. The next day, media was changed to the serum condition required, and cells were grown for 48 hours. Imaging was performed the following day. The two imaging protocols are summarized in Figure 1. For radioluminescence experiments, cells were incubated with FLT (18 MBq/mL for 60 min) or FDG (9 MBq/mL for 60 minutes after 30 min glucose fasting). The cells were then washed 3× with PBS, and a 500 mm-thick scintillator (CdWO4; two-face polished; MTI Corp., Richmond, CA) was placed on top of the cells. For FLT imaging, the cells were imaged using radioluminescence microscopy with a 15 min total exposure time, split into 12,000 frames (75 ms/frame). For FDG imaging, the total exposure time was 50 min, split into 30,000 frames (100 ms/frame). For fluorescence experiments, an EdU imaging kit (Life Technologies, C10337) and corresponding protocol was used (20 mM EdU for 1 hour at 37°C).
Sengupta D, & Pratx G P.r.o.f. (2016). Single-cell characterization of FLT uptake with radioluminescence microscopy. Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 57(7), 1136-1140.
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5

Proliferation Assays of COAD Cells

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For CCK‐8 (Beyotime) assay, the COAD cells (5000 cells) were seeded in a 96‐well plate, and 100 μL culture media supplemented with 10% CCK8 was added to each well and incubated at different times (12, 24, 36 and 48 hours). The absorbance (450 nm) was measured using microplate reader (Multiscan FC; Thermo Scientific). For EdU assay, EdU imaging kit (Life Technologies) was used to measure the DNA synthesis of COAD cells grown. EdU assay was carried out according to the reagent manufacturer's instructions, and immunostaining and EdU results were visualized using Leica DMI3000B microscope. We counted the positive cells.
Ni X., Ding Y., Yuan H., Shao J., Yan Y., Guo R., Luan W, & Xu M. (2019). Long non‐coding RNA ZEB1‐AS1 promotes colon adenocarcinoma malignant progression via miR‐455‐3p/PAK2 axis. Cell Proliferation, 53(1), e12723.
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6

Macrophage-Mediated Hepatocyte Proliferation

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Primary mouse hepatocytes were isolated from WT mice. Ly6ChiCX3CR1lo macrophages and Ly6CloCX3CR1hi macrophages were isolated at the indicated time points. Conditioned medium (CM) was collected from 2 × 105 Ly6ChiCX3CR1lo or Ly6CloCX3CR1hi macrophages, filtered through a 0.22 μm filter, and added to 1 × 104 hepatocytes. Hepatocytes were treated with macrophage-derived CM or HGF (50 ng/ml, Peprotech) for 12 h, followed by EdU (5-ethynyl-2′-deoxyuridine, 20 μM) pulsing for an additional 36 h. In another series of experiments, isolated hepatocytes were treated with Ly6CloCX3CR1hi macrophage-derived CM supplemented with or without the c-Met kinase inhibitor PHA665752 (2.5 μM, R&D systems). Hepatocytes undergoing DNA synthesis were visualized using the EdU Imaging Kit (Life Technologies). Imaging was performed using Olympus IX71 inverted fluorescence microscopes and EdU-positive cells were quantified by ImageJ software.
Yang W., Zhao X., Tao Y., Wu Y., He F, & Tang L. (2019). Proteomic analysis reveals a protective role of specific macrophage subsets in liver repair. Scientific Reports, 9, 2953.
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7

Evaluating Melanoma Cell Proliferation

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For cell counting kit-8 (CCK-8, Beyotime, Shanghai, China) assay, the transfected melanoma cells (5000 cells) were seeded in a 96-well plate, and the process is carried out as described previously [24 ]. Microplate reader (Multiscan FC, Thermo Scientific) was used to measure the absorbance at an optical density of 450 nm. For EdU assay, the DNA synthesis of melanoma cells grown was measured by using a EdU imaging kit (life Technologies, USA). The assay were carried out according to the manufacturer’s instructions. Immunostaining were visualized by using Leica DMI3000B microscope, and the positive cells were counted.
Luan W., Ding Y., Yuan H., Ma S., Ruan H., Wang J., Lu F, & Bu X. (2020). Long non-coding RNA LINC00520 promotes the proliferation and metastasis of malignant melanoma by inducing the miR-125b-5p/EIF5A2 axis. Journal of Experimental & Clinical Cancer Research : CR, 39, 96.
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8

Melanoma Cell Proliferation Assay

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Cells were seeded and cultured for 24 hours before transfection. An EdU imaging kit (Life Technologies, USA) was used to detect DNA synthesis in melanoma cells. Cell immunostaining was observed with a fluorescence microscope, and the cells positive for EdU staining were counted.
Xie Y, & Chen G. (2022). LncRNA SNHG12 promotes the malignant progression of melanoma by targeting miR-199b. Annals of Translational Medicine, 10(4), 226.
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9

Hepatocyte Proliferation Assay with Immune Cells

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Primary mouse hepatocytes were isolated from WT mice. Neutrophils and distinct macrophage subsets were isolated at the indicated time points. For co-culture studies, isolated hepatocytes were plated at a density of 1 × 105 cells ml−1, seeded with 2 × 106 cells ml−1 macrophages. Culture conditions consisted of RPMI1640 (Invitrogen) supplemented with 10% FBS, streptomycin (100 U ml−1) and penicillin (100 U ml−1). Conditioned medium (CM) was collected from 200,000 neutrophils or distinct macrophage subsets, filtered through a 0.22 μm filter, and added to 10,000 hepatocytes. Hepatocytes were treated with neutrophil or macrophage-derived CM for 12 h, followed by EdU (5-ethynyl-2′-deoxyuridine, 20 μM) pulsing for an additional 36 h. Hepatocytes undergoing DNA synthesis were visualized using the EdU Imaging Kit (Life Technologies). Imaging was performed using Olympus IX71 inverted fluorescence microscopes and EdU-positive cells were quantified by ImageJ software.
Yang W., Tao Y., Wu Y., Zhao X., Ye W., Zhao D., Fu L., Tian C., Yang J., He F, & Tang L. (2019). Neutrophils promote the development of reparative macrophages mediated by ROS to orchestrate liver repair. Nature Communications, 10, 1076.
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10

Cell Growth Assay and Proliferation Analysis

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For cell growth assays, H23/C18 and H23 cells pretreated with Dox or vehicle were seeded in 24 well plates at a density of 6x104 cells/well. Cells were then counted daily using a Z1 coulter particle counter (Beckman Coultier, Brea, CA) up to day 5. To evaluate proliferation of H23 and H23/C18 cells, 5-ethynyl-2′-deoxyuridine (EdU; 10 μM) was added to the culture medium 1 hr before fixation with 4% PFA. EdU incorporation was detected with the EdU Imaging Kit (Life Technologies; #10337).
Luo J., Chimge N.O., Zhou B., Flodby P., Castaldi A., Firth A.L., Liu Y., Wang H., Yang C., Marconett C.N., Crandall E.D., Offringa I.A., Frenkel B, & Borok Z. (2018). CLDN18.1 attenuates malignancy and related signaling pathways of lung adenocarcinoma in vivo and in vitro. International journal of cancer, 143(12), 3169-3180.
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