Journal: iScience

Article Title: OpenEMMU: A versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics

doi: 10.1016/j.isci.2025.113380

Figure Lengend Snippet: Application of OpenEMMU to normal and cancer cell lines for analysis of DNA replication (A) Diagram outlining the cell cycle phases, highlighting the S phase where thymidine analogs are incorporated during DNA replication. (B) Graph depicting the annual number of PUBMED articles mentioning BrdU or EdU. (C) Illustration of the EdU molecule and CuAAC reaction in cells that have incorporated EdU into nascent DNA. (D) Flow cytometry results show the proportion of EdU-labeled cells after a 2-h pulse in indicated cell lines ( n = 3). (E) Relationship between EdU fluorescence intensity and the proportion of EdU+ cells at varying EdU concentrations in hiPSCs ( n = 3). (F) Comparison of EdU and DNA fluorescence intensity at different EdU concentrations from (E). (G) Flow cytometry analysis of hiPSCs following a 1-h EdU pulse. The dotted lines indicate the gating strategy employed for both FACS and subsequent confocal microscopy of sorted cells (right panels) ( n = 3). (H) Confocal microscopy images of indicated EdU+ cancer cell lines after a 2-h EdU pulse, with α-Tubulin immunolabeling and DNA staining ( n = 3). E: Early DNA replication (S-phase); M: Mid DNA replication; L: Late DNA replication. (I) Cost per 0.5 mL reaction comparing OpenEMMU against two commercially available kit options (Invitrogen, kit 1; Vector Labs, kit 2).

Article Snippet: For aphidicolin-mediated DNA replication stress experiments, 6 hpf embryos were screened and incubated with 100 μM aphidicolin (MedChemExpress, HY-N6733) in 1% DMSO (v/v) for 20 h at 28°C in E3 medium.

Techniques: Flow Cytometry, Labeling, Fluorescence, Comparison, Confocal Microscopy, Immunolabeling, Staining, Plasmid Preparation

Journal: iScience

Article Title: OpenEMMU: A versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics

doi: 10.1016/j.isci.2025.113380

Figure Lengend Snippet: Compatibility of OpenEMMU for multi-parametric analysis of DNA replication in mouse and human immune cells (A) Outline of adult mice EdU injection and OpenEMMU protocol for studying DNA synthesis in various tissues and cells. (B) Flow cytometry of EdU signal using isolated bone marrow cells ( n = 3). (C) Flow cytometry of EdU signal using isolated splenocytes, highlighting percentages of rare DNA replicating cells (CD11b+ and CD11c+) ( n = 3). (D) Outline of freshly isolated human peripheral blood mononuclear cells (PBMCs) evaluation for EdU incorporation and responses to various immunological stimuli. (E) Gating strategy employed to detect DNA replication in activated human T-cells. The dashed lines indicate the selected gates. (F) Cell stimulation with activating antibodies or exposure to infectious disease-relevant immune stimulants showing the quantification of EdU+ DNA replicating CD3 + CD4 + CD25 + cells (dashed lines) ( n = 2).

Article Snippet: For aphidicolin-mediated DNA replication stress experiments, 6 hpf embryos were screened and incubated with 100 μM aphidicolin (MedChemExpress, HY-N6733) in 1% DMSO (v/v) for 20 h at 28°C in E3 medium.

Techniques: Injection, DNA Synthesis, Flow Cytometry, Isolation, Cell Stimulation

Journal: iScience

Article Title: OpenEMMU: A versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics

doi: 10.1016/j.isci.2025.113380

Figure Lengend Snippet: OpenEMMU for multiplexed imaging and DNA replication studies in the adult intestinal stem cell niche (A) OpenEMMU compatibility with ethyl cinnamate tissue clearing and 3D confocal laser imaging, during analysis of DNA synthesis in the small intestine (ileum) of adult mice injected with EdU for 4 h. Several proliferating cell regions are observed, including the adult stem cell crypt and the transient amplifying (TA) cells in the villi, highlighted by the dotted line. Asterisks indicate mitotic cells. (B) FFPE-processed ileum sections and confocal laser imaging of EdU in DNA-replicating cells, along with Ki67 and p-HH3 S10 that are EdU positive, visualized by z stack confocal microscopy. Dotted strokes highlight EdU + cells that are Ki67 negative. Asterisks indicate mitotic cells. (C) FFPE-processed ileum section, visualized using tiling confocal microscopy. The section underwent two cycles of 4-plex staining, with OpenEMMU used in the first cycle and IBEX applied in the second ( n = 3).

Article Snippet: For aphidicolin-mediated DNA replication stress experiments, 6 hpf embryos were screened and incubated with 100 μM aphidicolin (MedChemExpress, HY-N6733) in 1% DMSO (v/v) for 20 h at 28°C in E3 medium.

Techniques: Imaging, DNA Synthesis, Injection, Confocal Microscopy, Staining

Journal: iScience

Article Title: OpenEMMU: A versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics

doi: 10.1016/j.isci.2025.113380

Figure Lengend Snippet: Embryo and organ-specific analysis of DNA replication and mitosis in developing mouse embryos (A) FFPE-processed embryonic day E14.5 mouse embryo and confocal tile imaging of EdU signal in DNA replicating cells multiplexed with Ki67, αSMA, phospho-histone H3 S10 , and DNA labeling. αSMA immunolabelling depicts the developing musculature, heart cardiomyocytes, as well as smooth muscle cells in multiple organs and tissues ( n = 3). (B) Insets (B) show the magnification of the boxed areas using z stack confocal microscopy (a-i). (C) FFPE-processed E14.5 developing heart and confocal tile imaging of EdU signal in DNA replicating cells multiplexed with Ki67, αSMA, and DNA labeling ( n = 2). αSMA immunolabelling depicts the developing musculature, heart cardiomyocytes, and smooth muscle cells in multiple cardiac regions. (D) Insets (D) show the magnification of the boxed areas using z stack confocal microscopy (j-m). (E) E14.5 heart section underwent two 4-plex staining rounds: first with conjugated antibodies and WGA, then with OpenEMMU and IBEX ( n = 3).

Article Snippet: For aphidicolin-mediated DNA replication stress experiments, 6 hpf embryos were screened and incubated with 100 μM aphidicolin (MedChemExpress, HY-N6733) in 1% DMSO (v/v) for 20 h at 28°C in E3 medium.

Techniques: Imaging, DNA Labeling, Confocal Microscopy, Staining

Journal: iScience

Article Title: OpenEMMU: A versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics

doi: 10.1016/j.isci.2025.113380

Figure Lengend Snippet: DNA replication in self-organizing 3D human cardiac organoids (A) Protocol outline to generate self-organizing and beating hiPSC-derived human cardiac organoids (hCO), showing two distinct imaging strategies: FFPE organoid confocal imaging (a’) and 3D light sheet microscopy (b’). (B) FFPE-processed day 12 organoid and confocal tile imaging of EdU fluorescence in DNA replicating cells ( n = 6). NKX2-5 and cTnT expressing cardiac progenitors are highlighted in the boxed area. NKX2-5 and EdU double-positive cells are also shown (∗, magnified box). (C) Whole-mount light sheet microscopy (LSFM) 3D visualization of a hCO at day 19 showing EdU positive cells and cTnT expressing cells. DNA-labelled nuclei are also shown ( n = 6).

Article Snippet: For aphidicolin-mediated DNA replication stress experiments, 6 hpf embryos were screened and incubated with 100 μM aphidicolin (MedChemExpress, HY-N6733) in 1% DMSO (v/v) for 20 h at 28°C in E3 medium.

Techniques: Derivative Assay, Imaging, Microscopy, Fluorescence, Expressing

Journal: iScience

Article Title: OpenEMMU: A versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics

doi: 10.1016/j.isci.2025.113380

Figure Lengend Snippet: Analysis of DNA replication in whole zebrafish larvae (A) Danio rerio 5dpf larvae immersion protocol aimed at labeling DNA synthesis with EdU for 2h using FFPE (B) or whole-mount staining (D–F). (B) FFPE-processed larva sagittal cross section and confocal tile imaging of EdU signal in DNA replicating cells ( n = 3). αSMA-Cy3 immunolabeling depicts the developing musculature and growing myotomes. Insets (below) show the magnification of the boxed areas (a’-c’). Arrowhead shows a mitotic event. (C and D) Depigmentation and DEEP-Clear protocol allow the whole-mount visualization of growing larvae ( n = 6). (E and F) EdU and DNA labeling revealing highly proliferative cells, visualized by tiling (E) and z stack (F) confocal microscopy. (F) Insets (F) show the magnification of the boxed areas (a’-e’). CMZ, ciliary marginal zone; MTJ, myotendinous junction; nc, notochord; ms, myoseptum; mf, myofiber; bv, blood vessels.

Article Snippet: For aphidicolin-mediated DNA replication stress experiments, 6 hpf embryos were screened and incubated with 100 μM aphidicolin (MedChemExpress, HY-N6733) in 1% DMSO (v/v) for 20 h at 28°C in E3 medium.

Techniques: Labeling, DNA Synthesis, Staining, Imaging, Immunolabeling, DNA Labeling, Confocal Microscopy

Journal: iScience

Article Title: OpenEMMU: A versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics

doi: 10.1016/j.isci.2025.113380

Figure Lengend Snippet: Global DNA replication stress impairs zebrafish embryogenesis (A) Schematic representation of the experimental workflow. Danio rerio embryos were treated with aphidicolin, a DNA polymerase inhibitor, starting at 6 h postfertilization (hpf) to inhibit embryo-wide DNA replication during S-phase. (B) Bright-field images of representative embryos collected at 26 hpf, shown with the chorion intact (top) and removed (bottom) ( n = 10). (C) To assess DNA synthesis and cell proliferation under replication stress, dechorionated embryos were immersed in EdU for 1 h during aphidicolin treatment, followed by fixation and OpenEMMU. (D) Whole-mount imaging of depigmented and optically cleared embryos enabled the high-resolution visualization of proliferative activity (EdU incorporation) across embryonic tissues ( n = 10). Confocal z stack images show EdU incorporation, phospho-Histone H3 (Ser10) staining (pHH3) for mitotic cells, and nuclear DNA labeling. Insets (d′–d″) highlight magnified views of the boxed regions. The dotted lines indicate the developing myotomes, which are forming into segmented blocks of muscle.

Article Snippet: For aphidicolin-mediated DNA replication stress experiments, 6 hpf embryos were screened and incubated with 100 μM aphidicolin (MedChemExpress, HY-N6733) in 1% DMSO (v/v) for 20 h at 28°C in E3 medium.

Techniques: DNA Synthesis, Imaging, Activity Assay, Staining, DNA Labeling

Journal: iScience

Article Title: OpenEMMU: A versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics

doi: 10.1016/j.isci.2025.113380

Figure Lengend Snippet: Application of OpenEMMU to normal and cancer cell lines for analysis of DNA replication (A) Diagram outlining the cell cycle phases, highlighting the S phase where thymidine analogs are incorporated during DNA replication. (B) Graph depicting the annual number of PUBMED articles mentioning BrdU or EdU. (C) Illustration of the EdU molecule and CuAAC reaction in cells that have incorporated EdU into nascent DNA. (D) Flow cytometry results show the proportion of EdU-labeled cells after a 2-h pulse in indicated cell lines ( n = 3). (E) Relationship between EdU fluorescence intensity and the proportion of EdU+ cells at varying EdU concentrations in hiPSCs ( n = 3). (F) Comparison of EdU and DNA fluorescence intensity at different EdU concentrations from (E). (G) Flow cytometry analysis of hiPSCs following a 1-h EdU pulse. The dotted lines indicate the gating strategy employed for both FACS and subsequent confocal microscopy of sorted cells (right panels) ( n = 3). (H) Confocal microscopy images of indicated EdU+ cancer cell lines after a 2-h EdU pulse, with α-Tubulin immunolabeling and DNA staining ( n = 3). E: Early DNA replication (S-phase); M: Mid DNA replication; L: Late DNA replication. (I) Cost per 0.5 mL reaction comparing OpenEMMU against two commercially available kit options (Invitrogen, kit 1; Vector Labs, kit 2).

Article Snippet: E: Early DNA replication (S-phase); M: Mid DNA replication; L: Late DNA replication. (I) Cost per 0.5 mL reaction comparing OpenEMMU against two commercially available kit options (Invitrogen, kit 1; Vector Labs, kit 2).

Techniques: Flow Cytometry, Labeling, Fluorescence, Comparison, Confocal Microscopy, Immunolabeling, Staining, Plasmid Preparation

Journal: iScience

Article Title: OpenEMMU: A versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics

doi: 10.1016/j.isci.2025.113380

Figure Lengend Snippet: Compatibility of OpenEMMU for multi-parametric analysis of DNA replication in mouse and human immune cells (A) Outline of adult mice EdU injection and OpenEMMU protocol for studying DNA synthesis in various tissues and cells. (B) Flow cytometry of EdU signal using isolated bone marrow cells ( n = 3). (C) Flow cytometry of EdU signal using isolated splenocytes, highlighting percentages of rare DNA replicating cells (CD11b+ and CD11c+) ( n = 3). (D) Outline of freshly isolated human peripheral blood mononuclear cells (PBMCs) evaluation for EdU incorporation and responses to various immunological stimuli. (E) Gating strategy employed to detect DNA replication in activated human T-cells. The dashed lines indicate the selected gates. (F) Cell stimulation with activating antibodies or exposure to infectious disease-relevant immune stimulants showing the quantification of EdU+ DNA replicating CD3 + CD4 + CD25 + cells (dashed lines) ( n = 2).

Article Snippet: E: Early DNA replication (S-phase); M: Mid DNA replication; L: Late DNA replication. (I) Cost per 0.5 mL reaction comparing OpenEMMU against two commercially available kit options (Invitrogen, kit 1; Vector Labs, kit 2).

Techniques: Injection, DNA Synthesis, Flow Cytometry, Isolation, Cell Stimulation

Journal: iScience

Article Title: OpenEMMU: A versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics

doi: 10.1016/j.isci.2025.113380

Figure Lengend Snippet: OpenEMMU for multiplexed imaging and DNA replication studies in the adult intestinal stem cell niche (A) OpenEMMU compatibility with ethyl cinnamate tissue clearing and 3D confocal laser imaging, during analysis of DNA synthesis in the small intestine (ileum) of adult mice injected with EdU for 4 h. Several proliferating cell regions are observed, including the adult stem cell crypt and the transient amplifying (TA) cells in the villi, highlighted by the dotted line. Asterisks indicate mitotic cells. (B) FFPE-processed ileum sections and confocal laser imaging of EdU in DNA-replicating cells, along with Ki67 and p-HH3 S10 that are EdU positive, visualized by z stack confocal microscopy. Dotted strokes highlight EdU + cells that are Ki67 negative. Asterisks indicate mitotic cells. (C) FFPE-processed ileum section, visualized using tiling confocal microscopy. The section underwent two cycles of 4-plex staining, with OpenEMMU used in the first cycle and IBEX applied in the second ( n = 3).

Article Snippet: E: Early DNA replication (S-phase); M: Mid DNA replication; L: Late DNA replication. (I) Cost per 0.5 mL reaction comparing OpenEMMU against two commercially available kit options (Invitrogen, kit 1; Vector Labs, kit 2).

Techniques: Imaging, DNA Synthesis, Injection, Confocal Microscopy, Staining

Journal: iScience

Article Title: OpenEMMU: A versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics

doi: 10.1016/j.isci.2025.113380

Figure Lengend Snippet: Embryo and organ-specific analysis of DNA replication and mitosis in developing mouse embryos (A) FFPE-processed embryonic day E14.5 mouse embryo and confocal tile imaging of EdU signal in DNA replicating cells multiplexed with Ki67, αSMA, phospho-histone H3 S10 , and DNA labeling. αSMA immunolabelling depicts the developing musculature, heart cardiomyocytes, as well as smooth muscle cells in multiple organs and tissues ( n = 3). (B) Insets (B) show the magnification of the boxed areas using z stack confocal microscopy (a-i). (C) FFPE-processed E14.5 developing heart and confocal tile imaging of EdU signal in DNA replicating cells multiplexed with Ki67, αSMA, and DNA labeling ( n = 2). αSMA immunolabelling depicts the developing musculature, heart cardiomyocytes, and smooth muscle cells in multiple cardiac regions. (D) Insets (D) show the magnification of the boxed areas using z stack confocal microscopy (j-m). (E) E14.5 heart section underwent two 4-plex staining rounds: first with conjugated antibodies and WGA, then with OpenEMMU and IBEX ( n = 3).

Article Snippet: E: Early DNA replication (S-phase); M: Mid DNA replication; L: Late DNA replication. (I) Cost per 0.5 mL reaction comparing OpenEMMU against two commercially available kit options (Invitrogen, kit 1; Vector Labs, kit 2).

Techniques: Imaging, DNA Labeling, Confocal Microscopy, Staining

Journal: iScience

Article Title: OpenEMMU: A versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics

doi: 10.1016/j.isci.2025.113380

Figure Lengend Snippet: DNA replication in self-organizing 3D human cardiac organoids (A) Protocol outline to generate self-organizing and beating hiPSC-derived human cardiac organoids (hCO), showing two distinct imaging strategies: FFPE organoid confocal imaging (a’) and 3D light sheet microscopy (b’). (B) FFPE-processed day 12 organoid and confocal tile imaging of EdU fluorescence in DNA replicating cells ( n = 6). NKX2-5 and cTnT expressing cardiac progenitors are highlighted in the boxed area. NKX2-5 and EdU double-positive cells are also shown (∗, magnified box). (C) Whole-mount light sheet microscopy (LSFM) 3D visualization of a hCO at day 19 showing EdU positive cells and cTnT expressing cells. DNA-labelled nuclei are also shown ( n = 6).

Article Snippet: E: Early DNA replication (S-phase); M: Mid DNA replication; L: Late DNA replication. (I) Cost per 0.5 mL reaction comparing OpenEMMU against two commercially available kit options (Invitrogen, kit 1; Vector Labs, kit 2).

Techniques: Derivative Assay, Imaging, Microscopy, Fluorescence, Expressing

Journal: iScience

Article Title: OpenEMMU: A versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics

doi: 10.1016/j.isci.2025.113380

Figure Lengend Snippet: Analysis of DNA replication in whole zebrafish larvae (A) Danio rerio 5dpf larvae immersion protocol aimed at labeling DNA synthesis with EdU for 2h using FFPE (B) or whole-mount staining (D–F). (B) FFPE-processed larva sagittal cross section and confocal tile imaging of EdU signal in DNA replicating cells ( n = 3). αSMA-Cy3 immunolabeling depicts the developing musculature and growing myotomes. Insets (below) show the magnification of the boxed areas (a’-c’). Arrowhead shows a mitotic event. (C and D) Depigmentation and DEEP-Clear protocol allow the whole-mount visualization of growing larvae ( n = 6). (E and F) EdU and DNA labeling revealing highly proliferative cells, visualized by tiling (E) and z stack (F) confocal microscopy. (F) Insets (F) show the magnification of the boxed areas (a’-e’). CMZ, ciliary marginal zone; MTJ, myotendinous junction; nc, notochord; ms, myoseptum; mf, myofiber; bv, blood vessels.

Article Snippet: E: Early DNA replication (S-phase); M: Mid DNA replication; L: Late DNA replication. (I) Cost per 0.5 mL reaction comparing OpenEMMU against two commercially available kit options (Invitrogen, kit 1; Vector Labs, kit 2).

Techniques: Labeling, DNA Synthesis, Staining, Imaging, Immunolabeling, DNA Labeling, Confocal Microscopy

Journal: iScience

Article Title: OpenEMMU: A versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics

doi: 10.1016/j.isci.2025.113380

Figure Lengend Snippet: Global DNA replication stress impairs zebrafish embryogenesis (A) Schematic representation of the experimental workflow. Danio rerio embryos were treated with aphidicolin, a DNA polymerase inhibitor, starting at 6 h postfertilization (hpf) to inhibit embryo-wide DNA replication during S-phase. (B) Bright-field images of representative embryos collected at 26 hpf, shown with the chorion intact (top) and removed (bottom) ( n = 10). (C) To assess DNA synthesis and cell proliferation under replication stress, dechorionated embryos were immersed in EdU for 1 h during aphidicolin treatment, followed by fixation and OpenEMMU. (D) Whole-mount imaging of depigmented and optically cleared embryos enabled the high-resolution visualization of proliferative activity (EdU incorporation) across embryonic tissues ( n = 10). Confocal z stack images show EdU incorporation, phospho-Histone H3 (Ser10) staining (pHH3) for mitotic cells, and nuclear DNA labeling. Insets (d′–d″) highlight magnified views of the boxed regions. The dotted lines indicate the developing myotomes, which are forming into segmented blocks of muscle.

Article Snippet: E: Early DNA replication (S-phase); M: Mid DNA replication; L: Late DNA replication. (I) Cost per 0.5 mL reaction comparing OpenEMMU against two commercially available kit options (Invitrogen, kit 1; Vector Labs, kit 2).

Techniques: DNA Synthesis, Imaging, Activity Assay, Staining, DNA Labeling