JoVE Encyclopedie van Experimenten
Kankeronderzoek
0 weergaven • 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.
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