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Click it edu flow cytometry kit

Manufactured by Thermo Fisher Scientific
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The Click-iT EdU Flow Cytometry Kit is a tool used to detect and quantify cellular proliferation. It utilizes a thymidine analog, EdU (5-ethynyl-2'-deoxyuridine), which is incorporated into newly synthesized DNA during cell division. The kit provides the necessary reagents and protocols to label, detect, and analyze cell proliferation using flow cytometry.

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Lab products found in correlation

Cell light edu dna cell proliferation kit, by RiboBio (2 mentions) Mitotracker green fm, by Thermo Fisher Scientific (1 mentions) Anti cd49b, by Miltenyi Biotec (1 mentions) Anti f4 80, by Miltenyi Biotec (1 mentions) Anti cd45, by Miltenyi Biotec (1 mentions) Anti cd11c, by Miltenyi Biotec (1 mentions) Anti cd3, by Miltenyi Biotec (1 mentions) Facscanto 2 flow cytometer, by BD (1 mentions) Annexin 5 cy5, by BD (1 mentions) Bovine serum albumin (bsa), by GE Healthcare (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Lsr 2, by BD (1 mentions) Guava easycyte, by Merck Group (1 mentions) Facscalibur, by BD (1 mentions) Brdu flow cytometry kit, by BD (1 mentions) Cd45 microbeads, by Miltenyi Biotec (1 mentions) Facscanto 2, by BD (1 mentions) Fxcycle violet, by Thermo Fisher Scientific (1 mentions) Lsrii flow cytometer, by BD (1 mentions) Saponin, by Merck Group (1 mentions)

Spelling variants of this product

Flow cytometry (135 citations) Click-iT EdU (80 citations) Click-iTTM EdU Flow Cytometry Assay Kit (4 citations) Click-iTTM Plus EdU flow Cytometry Assay Kit (3 citations) Click-iTTM EdU Alexa FluorTM 647 Flow Cytometry Assay Kit (3 citations) Click-iTTM Plus EdU Alexa FluorTM 647 flow cytometry assay kit (2 citations) Click-iTTM EdU Pacific BlueTM Flow Cytometry Assay Kit (2 citations)

16 protocols using click it edu flow cytometry kit

1

Comprehensive C2C12 Myoblast Profiling

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Cell cycle/Edu experiments were performed by incubating growth phase C2C12 myoblast cultures with 10uM 5-ethynyl-2′-deoxyuridine (EdU) for 2 hours, fixing, counterstaining DNA with DAPI, and identifying Edu+ cells using a Click-IT Edu Flow Cytometry Kit (Thermo Fisher). Mitochondria were labeled in live cells using MitoTracker Green FM (Thermo Fisher) and analyzed by flow cytometry. Immune cell profiling was performed as follows: hindlimb muscles were enzymatically digested and filtered according to standard protocols (Bernet et al, 2014 ), cells were labeled with fluorescently conjugated primary antibodies according to manufacturer’s instructions, and flow cytometry performed on a MACSquant 10 analyzer. The following antibodies (Miltenyi Biotec) were used in this study: anti-CD45, anti-CD3, anti-CD49b, anti-CD11c, anti-MHC class II, anti-F4/80, anti-CD11c, and anti-GR1. The following is the gating strategy used to identify individual immune cell subtypes: NK: CD45+, CD3e−, CD49b+; Dendritic cell: CD45+, CD11c+, MHC class II+; M1 macrophage: CD45+, F4/80+, CD11b+, CD11c+; M2 macrophage: CD45+, F4/80+, CD11b+, CD11c−; Neutrophil: CD45+, CD11b+, GR1+; Pan T cells: CD45+, CD3e+; Cytotoxic T cells: CD45+, CD3e+, CD8+; Helper T cells: CD45+, CD3e+, CD4+; Regulatory T cells: CD45+, CD3e+, CD4+, CD25+. Samples were analyzed using either MACSquant software or FlowJo v10.
Hogan K.A., Cho D.S., Arneson P.C., Samani A., Palines P., Yang Y, & Doles J.D. (2017). Tumor-derived cytokines impair myogenesis and alter the skeletal muscle immune microenvironment. Cytokine, 107, 9-17.
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2

Quantifying Cell Death and Cycle

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For assessment of cell death, cells were harvested, washed, and stained with Annexin V-Cy5 according to the manufacturer’s instructions (BD Bioscience). For cell cycle analysis, cells were pulsed with EdU (10 μm) for 1 h, harvested and fixed with 4 % paraformaldehyde. Staining for EdU incorporation was carried out as indicated by the manufacturer’s instructions (Click-It EdU Flow Cytometry kit, Thermo Scientific) and the DNA content was visualized with propidium iodide. For all flow cytometry experiments, 10,000–50,000 cells were recorded. All samples were analyzed on a BD FACS Canto II flow cytometer. FlowJo v7.6.5. was used to process the obtained data.
Zurkirchen L., Varum S., Giger S., Klug A., Häusel J., Bossart R., Zemke M., Cantù C., Atak Z.K., Zamboni N., Basler K, & Sommer L. (2019). Yin Yang 1 sustains biosynthetic demands during brain development in a stage-specific manner. Nature Communications, 10, 2192.
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3

DNA Replication Monitoring by EdU

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Analysis of DNA replication by EdU incorporation was performed using the Click-iT EdU Flow Cytometry Kit (Thermo Fisher Scientific), following manufacturer’s instructions. Cells were pulsed with EdU at a final concentration of 10 μM for 30 minutes at 37 °C.
Neiman G., Scarafía M.A., La Greca A., Santín Velazque N.L., Garate X., Waisman A., Möbbs A.M., Kasai-Brunswick T.H., Mesquita F., Martire-Greco D., Moro L.N., Luzzani C., Bastos Carvalho A., Sevlever G.E., Campos de Carvalho A., Guberman A.S, & Miriuka S.G. (2019). Integrin alpha-5 subunit is critical for the early stages of human pluripotent stem cell cardiac differentiation. Scientific Reports, 9, 18077.
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4

Quantification of Cell Proliferation by EdU Flow Cytometry

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Click-iT EdU Flow Cytometry kit was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Cell proliferation was performed according to the manufacturer’s instructions23 (link). In brief, the cells were incubated with 5′-ethynyl-2′-deoxyuridine (EdU, 10 µM) for 2 h. Cells were harvested and mixed with 3 ml of PBS containing 1% bovine serum albumin (GE Healthcare Life Sciences), which was centrifuged at 1500 × g for 10 min at 4 °C and fixed with 100 µl 4% formaldehyde for 15 min. Cells were then washed and incubated with 100 µl saponin-based permeabilization buffer for 15 min. After permeabilization, the samples were incubated with CuSO4 and Alexa Flour® 488 coupled to azide for 30 min, and measured by the flow cytometry with 50,000 events.
Tao L., Yu H., Liang R., Jia R., Wang J., Jiang K, & Wang Z. (2019). Rev-erbα inhibits proliferation by reducing glycolytic flux and pentose phosphate pathway in human gastric cancer cells. Oncogenesis, 8(10), 57.
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5

Cell Cycle Analysis by Flow Cytometry

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Cells were cultured in 6-well plates and exposed to either 0.5 μM VX970 or DMSO. Cells were pulse-labelled with EdU for 1 hour prior to fixation in ice-cold 100% (v/v) ethanol and stored at -20°C until use. EdU was fluorescently labelled by conjugation to the Alexafluor-488 fluorophore using the Click-IT EdU Flow Cytometry Kit (Life Technologies) according to the manufacturer’s protocol. Total DNA content was assessed by incubation of cells for 15 minutes in 1.43 μM DAPI (Sigma) in PBS. Cell cycle profiles were generated using a BD LSR-II flow cytometer (BD Biosciences) and analysis performed using BD FACSDIVA software V8.01. Flow cytometry was performed according to the manufacturer’s guidelines.
Jones S.E., Fleuren E.D., Frankum J., Konde A., Williamson C.T., Krastev D.B., Pemberton H.N., Campbell J., Gulati A., Elliott R., Menon M., Selfe J.L., Brough R., Pettitt S.J., Niedzwiedz W., van der Graaf W.T., Shipley J., Ashworth A, & Lord C.J. (2017). ATR is a therapeutic target in synovial sarcoma. Cancer research, 77(24), 7014-7026.
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6

Hypoxic Stress and Cellular Response

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MSCs-mCherry and MSCs-Fstl1 were seeded in 6-well plates and incubated under hypoxic condition (94% N2, 5% CO2, and 1% O2) for 48 h. Cell death was measured by Annexin V-PE/7-AAD dead cell apoptosis kit (BD Pharmingen), and Annexin V-positive cells were quantified on a flow cytometry (Millipore Guava easyCyte) as described previously [11 (link), 12 (link)]. Cells treated with 25 μM etoposide for 24 h were used as positive controls for apoptosis assay. Cell proliferation was assessed by click-it EdU flow cytometry kit (Life Technologies) according to the manufacturer’s instructions. Briefly, cells were incubated with 10 μM ethynyldeoxyuridine (EdU) for 2 h immediately after hypoxic treatment. Nuclear EdU was further marked by binding of azide group of click-it®Alexa Fluor 647 fluorophore to alkyne group of EdU. EdU incorporation was finally analyzed on a flow cytometry (Millipore Guava easyCyte).
Shen H., Cui G., Li Y., Ye W., Sun Y., Zhang Z., Li J., Xu G., Zeng X., Zhang Y., Zhang W., Huang Z., Chen W, & Shen Z. (2019). Follistatin-like 1 protects mesenchymal stem cells from hypoxic damage and enhances their therapeutic efficacy in a mouse myocardial infarction model. Stem Cell Research & Therapy, 10, 17.
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7

Antibody-Dependent Cellular Phagocytosis Assay

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Effector cells were plated in 24 well plates, centrifuged to ensure an even distribution and incubated for 18 hours. Six hours prior to addition of target cells and opsonizing antibody, cells were treated with 25 ng/ml IFN-γ. Target cells were incubated with 5 μM 5-ethynyl-2′-deoxyuridine (EdU) provided with the Click iT EdU Flow Cytometry Kit (Life Technologies) for 48 hours prior to the assay, harvested by trypsinization, added at the appropriate effector:target ratios and incubated at 37°C for 3-6 hours in the presence of 1 μg/ml trastuzumab. The cells were harvested, treated with 10 μg/ml human IgG1 to block non-specific binding and incubated with 3 μg/ml PerCP-labeled anti-mouse CD45 antibody and 10 μg/ml Alexa 488-labeled pertuzumab for ten minutes on ice. The cells were subsequently washed, resuspended in 50% formalin in PBS and stained for EdU using the EdU flow cytometry kit protocol. Samples were analyzed using flow cytometry (BD FACSCalibur or LSR Fortessa). Percentage whole cell phagocytosis (WCP) was calculated as the fraction of EdU+ cells that were also CD45-positive and pertuzumab-negative.
Velmurugan R., Challa D.K., Ram S., Ober R.J, & Ward E.S. (2016). Macrophage-mediated trogocytosis leads to death of antibody-opsonized tumor cells. Molecular cancer therapeutics, 15(8), 1879-1889.
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8

Longitudinal BrdU and EdU Incorporation Analysis

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For longitudinal analysis of BrdU incorporation, mice were administered BrdU in their drinking water for up to 4 wk, the maximal possible duration due to BrdU toxicity, similar to what has previously been published for LCs (Merad et al., 2002 (link)). BrdU incorporation was detected using a BrdU Flow cytometry kit (BD Biosciences) following the manufacturer’s protocol. To quantify cells currently in S-phase, EdU incorporation assays were performed. Mice were injected i.p. with 1 mg of EdU and sacrificed 14 h later. Epidermal DETCs and LCs were enriched using CD45 microbeads (Miltenyi Biotec), and EdU incorporation was detected using the Click it EdU flow cytometry kit (Life Technologies) according to the manufacturer’s instructions.
Gentek R., Ghigo C., Hoeffel G., Jorquera A., Msallam R., Wienert S., Klauschen F., Ginhoux F, & Bajénoff M. (2018). Epidermal γδ T cells originate from yolk sac hematopoiesis and clonally self-renew in the adult. The Journal of Experimental Medicine, 215(12), 2994-3005.
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9

Cell Cycle Analysis via EdU Flow Cytometry

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Cell cycle analysis was performed with a Click-iT EdU Flow Cytometry Kit (Invitrogen; Thermo Fisher Scientific, Leiden NL). Cells were cultured with 50 µm 5-ethynyl-2-deoxyuridine (EdU) for 4 h and fixed and stained according to the manufacturers protocol for analysis on a BD FACS Canto II.
Wang Z., Coban B., Liao C.Y., Chen Y.J., Liu Q, & Danen E.H. (2023). GRHL2 Regulation of Growth/Motility Balance in Luminal versus Basal Breast Cancer. International Journal of Molecular Sciences, 24(3), 2512.
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10

Synchronization and Cell Cycle Analysis

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CEM T-ALL cells were synchronized in G1 phase, and then treated as indicated. Cells were pulsed with EdU 1 h before collection at different time points. Cells were fixed 4% paraformaldehyde, permeabilized with perm/wash reagent (Invitrogen), stained with Azide-AF647 (using click-chemistry, Invitrogen; Click-iT EdU Flow cytometry kit, #C10634) and FxCycle-Violet (Invitrogen), and then analyzed flow cytometry (a detailed description is available in the Supplementary Information).
Le T.M., Poddar S., Capri J.R., Abt E.R., Kim W., Wei L., Uong N.T., Cheng C.M., Braas D., Nikanjam M., Rix P., Merkurjev D., Zaretsky J., Kornblum H.I., Ribas A., Herschman H.R., Whitelegge J., Faull K.F., Donahue T.R., Czernin J, & Radu C.G. (2017). ATR inhibition facilitates targeting of leukemia dependence on convergent nucleotide biosynthetic pathways. Nature Communications, 8, 241.
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