Flow cytometry is most often used to study cells from the immune system. These cells isolated often from blood in the peripheral circulation, but can also be harvested from tissues. Cells found in tissues or organs are usually tightly attached to these sites, and this is a fundamental property of these cells that allows them to surveil the immune system and launch responses. However, additional cell preparation steps must be used in order to dissociate these cells from the tissue so they can be used for flow cytometry. Consider these technical aspects of using cells from the circulation or tissue as you plan your next flow cytometry experiment.

Peripheral Whole Blood

Whole blood is a rich source of immune cells and these cells can be measured directly by flow cytometry using whole blood assays or can be purified using density gradient centrifugation, which separates blood into components by size and density. Whole blood is layered on top of a density gradient medium such as a sucrose solution or a specialized solution like Ficoll in a test tube and undergoes centrifugation. Ficoll gradients are widely used for this separation technique as blood components are isolated into visible bands. The lowest density component is on top (plasma), under which appears a band of PBMCs (peripheral blood mononuclear cells, including lymphocytes), Ficoll, granulocytes, and a bottom pellet of red blood cells. The band of cells needed for flow cytometry can be collected and then washed and processed for staining with your flow cytometry panel or cryopreserved for later use.

Tissue - Mechanical versus Enzymatic Dissociation

Two different techniques are used to dissociate cells from tissue, mechanical dissociation or enzymatic dissociation.

Mechanical dissociation uses tools such as a tissue homogenizer, which can be a specialized mortar and pestle or a more sophisticated instrument, to grind up tissue and liberate cells. This brute force method can successfully isolate cells, but can cause cell damage and does not always result in high cell yields.

Enzymatic dissociation uses enzymes such as collagenase to break down protein components in tissue and detach cells. It can be completed in addition to mechanical separation or used alone. Enzymatic dissociation can alter cells because enzymes may cleave cell surface proteins, so you need to determine if your cells of interest are altered by such a treatment during a proof of concept study prior to analysis by Flow Cytometry.  

As you tackle your next flow cytometry project with blood or tissue samples, consider the wide array of techniques available to help you separate cells and optimize cell yields.