Single cells must be suspended at a density of 105 –107 cells/ml to keep the narrow bores of the flow cytometer and its tubing from clogging up. The concentration also influences the rate of flow sorting, which typically progresses at 2,000–20,000 cells/second. Higher sort speeds can result in lower yield or recovery
Phosphate buffered saline (PBS) is a common suspension buffer. The most straightforward samples for flow cytometry include nonadherent cells from culture, waterborne microorganisms, bacteria, and yeast. Even whole blood is easy to use — red cells are usually removed by a simple lysis step. It is then possible to quickly identify lymphocytes, granulocytes, and monocytes by their FSC and SSC characteristics (see Figure 15).
However, researchers may also wish to analyze cells from solid tissues, for example, liver or tumors. In order to produce single cells, the solid material must be disaggregated. This can be done either mechanically or enzymatically. Mechanical disaggregation is suitable for loosely bound structures such as adherent cells from culture, bone marrow, and lymphoid tissue. It involves passing a suspension of chopped tissue through a fine-gauge needle several times followed by grinding and sonication as necessary.
Enzymes are used to disrupt protein-protein interactions and the extracellular matrix that holds cells together. Their action depends on factors including pH, temperature, and cofactors, so care must be taken when choosing an enzyme. For example, pepsin works optimally between pH 1.5 and 2.5, but the acidic conditions would damage cells if left unneutralized for too long, and cell surface antigens of interest might be lost. Chelators like ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-bis(2-aminoethylether)- N,N,N',N'-tetraacetic acid (EGTA) can remove divalent cations responsible for maintaining cell function and integrity, but their presence may inhibit certain enzymes. For example, collagenase requires Ca2+ for activity. Optimizing the isolation of an epitope under investigation via disaggregation, either enzymatic or mechanical, is often a trial and error process.
To study intracellular components, for example, cytokines, by flow cytometry, the plasma membrane of the cell must be permeabilized to allow dyes or antibody molecules through while retaining the cell’s overall integrity. Low concentrations (up to 0.1%) of nonionic detergents like saponin are suitable. In summary, the method for sample preparation will depend on the starting material and the nature of the epitope. Although it is not possible to describe every method here, some standard protocols are provided in this chapter.
The Flow Cytometry protocols provided detail the procedures for the treatment and staining of cells prior to using a flow cytometer. Protocols are available for;
View all Flow Cytometry protocols
EDTA Ethylenediaminetetraacetic acid
EGTA Ethyleneglycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid
Chapter 3: Intracellular Antigens
Chapter 5: Troubleshooting
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