Retro-Orbital Blood Sampling: A Method for Isolating Mononuclear Cells from the Retro-Orbital Sinus of a Mouse

- 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.