In Vivo EdU Cell Proliferation Assay for Flow Cytometry

Product Information

Reliable, Non-Radioactive Detection of DNA Synthesis in Living Organisms

The In Vivo EdU Cell Proliferation Assay for Flow Cytometry provides a robust and non-radioactive method for tracking de novo DNA synthesis in living organisms. Unlike traditional BrdU or ³H-thymidine assays, this approach avoids harsh DNA denaturation and radioactive hazards, enabling fast, precise, and multiplex-compatible detection of proliferating cells in complex biological systems.

Principle of the Assay

EdU (5-ethynyl-2′-deoxyuridine), a thymidine analog, is administered to the organism via injection or oral dosing. During the S-phase, EdU is incorporated into replicating DNA. Detection of the incorporated EdU is achieved through a copper-catalyzed azide-alkyne cycloaddition (click chemistry), which conjugates EdU to a fluorescent azide dye without requiring DNA denaturation or antibodies. This mild and highly specific reaction preserves sample integrity and supports downstream multiplexing.

How it works:

1. In vivo EdU Incorporation

EdU can be delivered to animals (e.g., mice, rats, zebrafish) via intraperitoneal, intramuscular, or subcutaneous injection, orally, or through direct incubation, depending on the model. Inside cells, EdU is phosphorylated into EdU-triphosphate and integrated by DNA polymerase, marking actively dividing cells. Recommended dosing: 10 µM for short studies (2–24 hours) or 1–5 µM for longer labeling periods.

2. Cell Preparation

After EdU incubation (2 hours to 1 day), harvest cells or tissues, then fix with paraformaldehyde to preserve structure and permeabilize with 0,5% saponin solution to allow reagent access to the DNA, preparing samples for detection.

3. Click Chemistry Detection

A rapid click reaction conjugates EdU’s alkyne group in the DNA to a fluorescent azide dye using a copper catalyst, forming a stable triazole bond that makes dividing cells to emit a bright fluorescent signal with minimal background noise, detectable by flow cytometry.

4. Flow Cytometry Analysis

Analyze labeled cells using a flow cytometer with standard channels (488, 555, 594, or 647 nm). Apply gating strategies including forward/side scatter, live/dead discrimination, and marker-based identification for accurate interpretation.

To obtain more comprehensive datasets, EdU labeling can be combined with immunohistochemistry (IHC) and DNA counterstaining. Perform EdU detection prior to immunohistochemistry (IHC) to prevent antigen loss caused by the copper-catalyzed reaction. Add DAPI or antibody staining after the click reaction to enable multiplex imaging, with DNA counterstaining typically performed last. This sequencing allows collection of richer multiparametric data while minimizing spectral overlap.

Why Choose baseclick’s In Vivo EdU Assay?

• Superior Accuracy: Direct labeling of proliferating cells without antibodies or radiation.
• Fast and Efficient: Detection completed in ~30 minutes post-fixation.
• Flexible Delivery: Injection, oral dosing, or incubation for diverse models.
• Multiplex Ready: Combine with antibody staining or nuclear dyes for comprehensive analysis.
• Broad Applicability: Suitable for mammals, fish, insects, worms, and plants.

Key Differences between In Vivo and  In Vitro

The in vivo kit is designed for whole-organism studies, enabling systemic EdU uptake and tissue-specific analysis in models such as mice, zebrafish, or plants. It is ideal for drug efficacy, toxicology, and developmental biology research. In contrast, the in vitro kit is optimized for controlled cell culture experiments, supporting high-throughput screening and rapid drug testing. Both kits use the same reliable click chemistry and flow cytometry detection for consistent, high-quality results.

Advantages

The In Vivo EdU Cell Proliferation Assay offers significant advantages over traditional methods:

• Non-Radioactive and Safe: Eliminates hazards of ³H-thymidine.
• Gentle on Samples: No harsh DNA denaturation as required for BrdU.
• High Sensitivity and Specificity: Detects proliferating cells across diverse organisms.
• Rapid and Efficient: The EdU detection process takes approximately 30 minutes, streamlining workflows and saving valuable time.
• Versatile and Flexible: Available in 3 kit sizes and compatible.
• Multiplexing Capability: Compatible with immunostaining and nuclear dyes.

Kit Contents

• EdU (S, M, or L sizes)
• Fluorescent dye (488, 555, 594, or 647 nm)
• Click reaction reagents
• Fixation and permeabilization solutions
• Clear protocol for in vivo and flow cytometry workflows

Tips for Success

• Use 10 µM EdU for short-term labeling or 1–5 µM for extended studies.
• Store fixed samples at 4 °C for flexible analysis timing.
• Perform EdU detection before antibody staining to preserve epitopes.

LITERATURE

An efficient protocol for in vivo labeling of proliferating epithelial cells, C. Michel et al., 2018, J. Immunol. Methods, Vol. 457, p. 82-86.

https://doi.org/10.1016/j.jim.2018.03.015

Cisplatin-induced DNA double-strand breaks promote meiotic chromosome synapsis in PRDM9-controlled mouse hybrid sterility, L. Wang et al., 2018, eLife, Vol. 7, p. e42511.

https://doi.org/10.7554/eLife.42511

FXR Regulates Intestinal Cancer Stem Cell Proliferation, T. Fu et al., 2019, Cell, Vol. 176, p. 1098-1112.

https://doi.org/10.1016/j.cell.2019.01.036

A modified method to analyse cell proliferation using EdU labelling in large insect brains, A. Anton et.al., 2023, PLoS One, Vol. 18(10), p. e0292009.

PLoS One; doi: 10.1371/journal.pone.0292009. eCollection 2023.

EdU-Based Assay of Cell Proliferation and Stem Cell Quiescence in Skeletal Tissue Sections, M. Angelozzi et al., 2021, Methods in Molecular Biology, Vol. 2230, p. 357–365.

1007/978-1-0716-1028-2_21