Retro-Orbital Blood Sampling: A Method for Isolating Mononuclear Cells from the Retro-Orbital Sinus of a Mouse
JoVE Encyclopedia of Experiments
Cancer Research
0 views • 3:18 min • April 30th, 2023
- First, hold an anesthetized mouse by its neck and protrude the eye. Insert a fine capillary tube into the medial canthus of the eye. Allow the blood to flow by capillary action into a tube. Transfer the blood into an EDTA-coated tube and maintain it on ice. EDTA binds the calcium ions, preventing blood coagulation. Lower temperature reduces the glycolytic activity of cells and the chances for microbial contamination.
Next, dilute the blood with a buffer containing EDTA and mix well. Carefully add a layer of an appropriate separating solution to the bottom, without disturbing the blood. Centrifuge the suspension. The polymer in the separating solution aggregates the red blood cells and sediments at the bottom. Mononuclear cells and platelets, being lighter than the polymer, deposits above the polymeric gradient.
Remove the top layer containing plasma and collect the opaque white ring containing mononuclear cells. Dilute the cells using PBS and centrifuge to remove residual plasma and platelets. Use the isolated mononuclear cells for further analysis. The following protocol will demonstrate mononuclear cells' isolation from a congenic mouse injected with a leukemic cell line C1498.
- Insert a capillary tube into the medial canthus of the eye of the anesthetized leukemic animal. 100 to 200 microliters of blood will be drawn from the orbital sinus into the capillary tube. Remove the tube and control the bleeding with the gentle application of a sterile gauze sponge onto the eye. Then, transfer the blood into an EDTA tube and store the tube on ice until the mononuclear cell isolation.
To isolate the blood mononuclear cells, first add PBS supplemented with 1 millimolar EDTA up to a final volume of 500 microliters. Then, transfer the sample to a microcentrifuge tube. Next, use a 1-milliliter syringe equipped with a 30-gauge needle to carefully layer 500 microliters of separating solution under the blood, taking care not to mix the layers.
Separate the cells by centrifugation and use a pipette to transfer the opaque white mononuclear cell layer into a new microcentrifuge tube. Then wash immune cells in 1 milliliter of PBS. Centrifuge them and re-suspend the pellet in 600 microliters of FACS buffer for staining and analysis by flow cytometry.
Overview
This article details a protocol for isolating mononuclear cells from the blood of anesthetized mice, specifically those injected with a leukemic cell line. The method emphasizes the importance of maintaining sample integrity through temperature control and the use of EDTA to prevent coagulation.
Key Study Components
Area of Science
- Cell biology
- Hematology
- Oncology
Background
- Mononuclear cells play a crucial role in immune response.
- Isolation of these cells is essential for various analyses.
- EDTA is commonly used to prevent blood coagulation.
- Temperature control is vital to reduce microbial contamination.
Purpose of Study
- To provide a detailed protocol for isolating mononuclear cells.
- To demonstrate the procedure using a leukemic mouse model.
- To facilitate further analysis of immune cells.
Methods Used
- Blood collection from the orbital sinus using a capillary tube.
- Use of EDTA to prevent coagulation and maintain sample integrity.
- Centrifugation to separate mononuclear cells from plasma and platelets.
- Flow cytometry for analysis of isolated cells.
Main Results
- Successful isolation of mononuclear cells from mouse blood.
- Clear separation of cell layers during centrifugation.
- Preservation of cell viability for subsequent analysis.
- Protocol can be applied to other experimental models.
Conclusions
- The described method is effective for isolating mononuclear cells.
- Maintaining proper conditions is crucial for sample quality.
- This protocol can aid in the study of immune responses in leukemic models.
Frequently Asked Questions
Executive Industry Relevance
Reliable isolation of mononuclear cells from murine blood is foundational for immunophenotyping, mechanistic studies, and preclinical model validation in hematology and oncology pipelines. This protocol enables standardized sample acquisition and preparation, supporting reproducible immune cell analysis and translational research continuity. Its integration strengthens predictive confidence in disease-relevant systems and informs early-stage portfolio decisions.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of immune cell populations in disease models for mechanistic de-risking.
- Supports functional target validation by providing viable mononuclear cells for downstream assays.
- Facilitates predictive confidence in immune response characterization within preclinical studies.
Screening & Assay Development
- Provides standardized mononuclear cell preparations for assay development and optimization.
- Ensures reproducibility and quantitative consistency in immune cell-based screening workflows.
- Enables reliable evaluation of compound effects on primary immune cells in vitro.
Translational & Preclinical Research
- Aligns with disease-relevant murine models for translational biomarker studies.
- Maintains continuity from discovery through preclinical immune profiling and validation.
- Supports risk-adjusted advancement by enabling robust immune monitoring in vivo.
Pipeline & Workflow Integration
This method positions at the interface of in vivo model experimentation and ex vivo immune cell analysis, bridging early discovery and preclinical validation stages.
- Discovery Biology: Supports hypothesis testing and pathway clarification by enabling immune cell isolation from disease models.
- Screening: Delivers assay-ready mononuclear cells with reproducible yields for downstream applications.
- Analytics: Provides quantitative cell populations for flow cytometry and other analytical platforms.
- Translational Research: Facilitates biomarker alignment and immune monitoring in preclinical models.
- Enterprise Reuse: Establishes a reusable protocol for consistent immune cell isolation across studies and teams.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in immune cell studies.
- Operational Value: Standardizes sample handling, improving reproducibility and scalability across experiments.
- Strategic Value: Enables informed go/no-go decisions by providing robust immune cell data early in the pipeline.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of immunology and oncology assets.
Implementation Considerations
- Requires technical expertise in murine handling and blood collection techniques.
- Needs access to centrifugation and flow cytometry infrastructure for cell isolation and analysis.
- Demands cross-team standardization of sample processing protocols to ensure data comparability.
- Adaptation may be necessary for different mouse strains or disease models.
- Sample volume limitations and animal welfare considerations must be managed per protocol.
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Last updated: 29 August 2026