5 ethynyl 2 deoxyuridine (edu), by Cayman Chemical - Product details

1

Evaluating EpCAM CAR-T Cell Efficacy

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EpCAM CAR-modified or untransduced control thy1.1 + T cells (5 × 10⁶) were 1:1 mixed with thy1.2+ untransduced control T cells (5 × 10⁶), after which mixed T cells were intravenously injected into thy1.1+ thy1.2+ B6 mice. Three days later, peripheral blood was subjected to flow cytometry analysis to evaluate proportions of transferred T cells.
EGFRvIII-specific CAR-T cells containing the same intracellular signaling domains as EpCAM CAR-T were used as a control. 1 × 10⁷ thy1.2+ derived EGFRvIII CAR-T cells or EpCAM CAR-T cells were transferred into thy1.1+ B6 mice separately. Mice were euthanized on day 3 post T cell infusion, and spleen, colon, and lung were dissected, minced, and digested to obtain a single-cell suspension. All samples were incubated in ACK lysis buffer on ice for 30 sec. Numbers of transferred and recipient T cells in each mouse were analyzed by flow cytometry. To measure in vivo proliferation, 12 h before flow analysis, mice were intraperitoneally injected with 20 mg/kg 5-ethynyl-2´-deoxyuridine (EdU) (Cayman Chemical). Transferred CAR-T cell proliferation (indicated by EdU staining) was analyzed using a Click-iT Plus kit (Invitrogen™) according to the manufacturer’s instructions.

Qin D., Li D., Zhang B., Chen Y., Liao X., Li X., Alexander P.B., Wang Y, & Li Q.J. (2020). Potential lung attack and lethality generated by EpCAM-specific CAR-T cells in immunocompetent mouse models. Oncoimmunology, 9(1), 1806009.

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2

Quantifying Atherosclerotic Plaque Proliferation

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Mice were injected intraperitoneally with 50 mg kg⁻¹ of 5-Ethynyl-2′-deoxyuridine (EdU) (#61135-33-9, Cayman Chemicals, United States) in 100 μL volume 96 h before sacrifice over 16 weeks of HFD feeding. 4% PFA-fixed frozen aortic root sections with 5-μm-thickness were used, and EdU staining was performed using a Click & Go Cell reaction buffer kit with Alexa-Fluor 488 (#1263, Click chemistry tools, United States) following the manufacturer's instructions. Anti-Mac3 and anti-SMα-actin-cy3 were used to visualize the distribution of each cell type. Quantification of proliferation was performed by counting the numbers of EdU-positive nuclei in plaque area.

Jung I.H, & Stitziel N.O. (2024). Integrin α9β1 deficiency does not impact the development of atherosclerosis in mice. Heliyon, 10(4), e25760.

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3

Microbiota Reconstitution in Germ-Free Mice

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Mice received a combination of vancomycin, neomycin, ampicillin and metronidazole (Sigma) with the addition of 1% glucose for one-week ad libitum. Proliferation was assessed 2 hr after inter peritoneum injection with 5-Ethynyl-2′-deoxyuridine (Cayman). Cre recombineering in the Lgr5-EGFP-IRES-creERT2 mice was achieved after a single inter peritoneum injection with 200 μL tamoxifen (10mg/mL) (Sigma). Conventionalized (Conv-D) mice were generated by colonizing germ-free (GF) C57BL/6 mice with caecal/faecal microbiota from conventionally raised (Conv-R) C57BL/6 donors. For each experiment, caecal and colonic content was harvested from three ConvR donors under aseptic conditions. Caecal/faecal content was pooled in 10 mL PBS supplemented with 0.1% (w/v) cysteine (Sigma) and homogenized using a T10 ULTRA-TURRAX rotor stator homogenizer (IKA). Freshly prepared homogenate (200 μl) was immediately administered to GF mice via gavage, and the resulting Conv-D mice were subsequently housed under conventional conditions for the duration of the experiment. Female donor and recipient mice were used for all conventionalization experiments.

Matthews A.N., Friend D.S., Zimmermann N., Sarafi M.N., Luster A.D., Pearlman E., Wert S.E, & Rothenberg M.E. (1998). Eotaxin is required for the baseline level of tissue eosinophils. Proceedings of the National Academy of Sciences of the United States of America, 95(11), 6273-6278.

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4

Microbiota Reconstitution in Germ-Free Mice

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Mice received a combination of vancomycin, neomycin, ampicillin and metronidazole (Sigma) with the addition of 1% glucose for one-week ad libitum. Proliferation was assessed 2 hr after inter peritoneum injection with 5-Ethynyl-2′-deoxyuridine (Cayman). Cre recombineering in the Lgr5-EGFP-IRES-creERT2 mice was achieved after a single inter peritoneum injection with 200 μL tamoxifen (10mg/mL) (Sigma). Conventionalized (Conv-D) mice were generated by colonizing germ-free (GF) C57BL/6 mice with caecal/faecal microbiota from conventionally raised (Conv-R) C57BL/6 donors. For each experiment, caecal and colonic content was harvested from three ConvR donors under aseptic conditions. Caecal/faecal content was pooled in 10 mL PBS supplemented with 0.1% (w/v) cysteine (Sigma) and homogenized using a T10 ULTRA-TURRAX rotor stator homogenizer (IKA). Freshly prepared homogenate (200 μl) was immediately administered to GF mice via gavage, and the resulting Conv-D mice were subsequently housed under conventional conditions for the duration of the experiment. Female donor and recipient mice were used for all conventionalization experiments.

van der Post S., Birchenough G.M, & Held J.M. (2021). NOX1-dependent redox signaling potentiates colonic stem cell proliferation to adapt to the intestinal microbiota by linking EGFR and TLR activation. Cell reports, 35(1), 108949.

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5

Quantifying DNA Synthesis Dynamics

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If measuring 5-ethynyl-2'-deoxyuridine (EdU) incorporation, cells were pulsed with 50 μM EdU (Cayman Chemical Cat# 20518) in growth media for 8 min prior to fixation and pre-extraction, unless otherwise stated. EdU is incorporated throughout the EdU pulse, such that incorporated EdU reflects the average rate of DNA synthesis over the length of the pulse. Thus an 8 min short EdU pulse is more reflective of the instantaneous DNA synthesis rate compared to a longer pulse such as 1 h. After blocking cells (prior to primary antibodies), cells were washed once with PBS, and then a click reaction^{67 (link)} was performed in 2 mM CuSO₄, 20 mg/mL sodium ascorbate in TBS (Tris 50 mM, NaCl 150 mM pH 8.3) with 3 μM AFDye 488 picolyl azide (Click Chemistry Tools Cat# 1276) or AFDye 647 picolyl azide (Click Chemistry Tools Cat# 1300) for 30 min, followed by a PBS wash.

Ratnayeke N., Baris Y., Chung M., Yeeles J.T, & Meyer T. (2023). CDT1 inhibits CMG helicase in early S phase to separate origin licensing from DNA synthesis. Molecular cell, 83(1), 26-42.e13.

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6

Cell Cycle Analysis of Bcl2-Expressing K562 Cells

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The cell cycles of K562-Bcl2 cells, with or without Palbociclib, DPKi #1/2, and ATMi, were assessed based on DNA replication activity. Briefly, 50 μM EdU (Cayman #20518) was added to each condition and incubated for 30 minutes. The EdU-treated cells were fixed with 2% formaldehyde (EMS #15710) for 15 min at room temperature, permeabilized with 0.5% TritonX-100 for 15 min at room temperature and subjected to click labeling reaction using AFDye 488 Azide (Click Chemistry Tools, #1314). The AFDye 488-labeled cells were analyzed by flow cytometry using the FITC channel.

Wang J., Sadeghi C.A, & Frock R.L. (2024). DNA-PKcs suppresses illegitimate chromosome rearrangements. Nucleic Acids Research, 52(9), 5048-5066.

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7

Permanent Labeling of Cortical Neurons

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To permanently label newly generated cortical neurons, pregnant dams at E16.5 were single-dosed with 5–ethynyl–2′–deoxyuridine (EdU, Cayman Chem) in 1X PBS at 30mg/kg body weight via intraperitoneal injection. Pups were perfused at P14 and sectioned as described above. EdU detection was conducted upon the completion of immunofluorescence labeling. After washing in PBS for 10 minutes, brains sections were incubated with Alexa 647-conjugated Azide (Biotium) at 1.6μM, in solution containing 0.1M Tris-HCl (pH 8.8), 0.43 X PBS, 4 mM CuSO₄, and 0.1M Ascorbate, for 20 minutes. Brains sections were washed with PBS/T three times at 20 minutes each, stained with DAPI and mounted for confocal imaging.

Krupa O., Fragola G., Hadden-Ford E., Mory J.T., Liu T., Humphrey Z., Rees B.W., Krishnamurthy A., Snider W.D., Zylka M.J., Wu G., Xing L, & Stein J.L. (2021). NuMorph: Tools for cortical cellular phenotyping in tissue-cleared whole-brain images. Cell reports, 37(2), 109802.

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8

Myofiber Isolation and Proliferation

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The myofibers from both WT and Prmt5^MKO mice were isolated and cultured in a Petri dish (60 mm) coated with horse serum in DMEM supplemented with 20% FBS (HyClone), 4 ng/ml basic FGF (Promega), and 1% Penicillin-streptomycin (HyClone) at 37°C. EdU (Cayman Chemical) was added to the culture medium at a concentration of 1mM. Cultured single myofibers were sampled at the designated time points for analysis.

Kim K.H., Oprescu S.N., Snyder M.M., Kim A., Jia Z., Yue F, & Kuang S. (2023). PRMT5 mediates FoxO1 methylation and subcellular localization to regulate lipophagy in myogenic progenitors. Cell reports, 42(11), 113329.

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9

Quantifying DNA Synthesis Dynamics

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If measuring 5-ethynyl-2'-deoxyuridine (EdU) incorporation, cells were pulsed with 50 μM EdU (Cayman Chemical Cat# 20518) in growth media for 8 min prior to fixation and pre-extraction, unless otherwise stated. EdU is incorporated throughout the EdU pulse, such that incorporated EdU reflects the average rate of DNA synthesis over the length of the pulse. Thus an 8 min short EdU pulse is more reflective of the instantaneous DNA synthesis rate compared to a longer pulse such as 1 h. After blocking cells (prior to primary antibodies), cells were washed once with PBS, and then a click reaction^{67 (link)} was performed in 2 mM CuSO₄, 20 mg/mL sodium ascorbate in TBS (Tris 50 mM, NaCl 150 mM pH 8.3) with 3 μM AFDye 488 picolyl azide (Click Chemistry Tools Cat# 1276) or AFDye 647 picolyl azide (Click Chemistry Tools Cat# 1300) for 30 min, followed by a PBS wash.

Ratnayeke N., Baris Y., Chung M., Yeeles J.T, & Meyer T. (2023). CDT1 inhibits CMG helicase in early S phase to separate origin licensing from DNA synthesis. Molecular cell, 83(1), 26-42.e13.

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10

Leukemic Blast Cell Cycle Analysis

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Cell labeling was done by i.p. injection of mice with 50 mg/kg of freshly prepared EdU (Cayman Chemical Company, catalog no. 20518). After three to four hours, BM was harvested, and human cells were negatively selected by mouse cell depletion using mouse CD45 magnetic beads (Miltenyi). The human BM cells were then stained with CD45 and CD33 to identify the leukemic blast. Cell-cycle/proliferation analysis was performed using the Click-iT Plus EdU Flow Cytometry Assay Kit (Invitrogen, catalog no. C10420) following the manufacturers’ instructions. Fixable viability dye (BioLegend) was used to discriminate the death population. Single-color controls were used to set compensations, and fluorescence minus one control were used to set gates. Analysis was performed with FlowJo software.

Galán-Díez M., Borot F., Ali A.M., Zhao J., Gil-Iturbe E., Shan X., Luo N., Liu Y., Huang X.P., Bisikirska B., Labella R., Kurland I., Roth B.L., Quick M., Mukherjee S., Rabadán R., Carroll M., Raza A, & Kousteni S. (2022). Subversion of Serotonin Receptor Signaling in Osteoblasts by Kynurenine Drives Acute Myeloid Leukemia. Cancer Discovery, 12(4), 1106-1127.

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5 ethynyl 2 deoxyuridine (edu)

Lab products found in correlation

11 protocols using 5 ethynyl 2 deoxyuridine (edu)

Evaluating EpCAM CAR-T Cell Efficacy

Quantifying Atherosclerotic Plaque Proliferation

Microbiota Reconstitution in Germ-Free Mice

Microbiota Reconstitution in Germ-Free Mice

Quantifying DNA Synthesis Dynamics

Cell Cycle Analysis of Bcl2-Expressing K562 Cells

Permanent Labeling of Cortical Neurons

Myofiber Isolation and Proliferation

Quantifying DNA Synthesis Dynamics

Leukemic Blast Cell Cycle Analysis

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