Sample Preparation

Single cells must be suspended at a density of 10e5–10e7 cells/ml to prevent 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 2000–20,000 cells/second. However, higher sort speeds may decrease the purity of the preparation.

Phosphate buffered saline (PBS) is a common suspension buffer and the most straightforward samples for flow cytometry include non-adherent cells from culture, water-borne micro-organisms, 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/SSC characteristics.

However, researchers may also wish to analyze cells from solid tissues e.g. 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 e.g. 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 hold cells together. Their action is dependent on factors including pH, temperature and co-factors, so care must be taken when choosing an enzyme. For example, pepsin works optimally between pH 1.5–2.5 but the acidic conditions would damage cells if left unneutralized for too long, and cell surface antigens of interest may be lost. Chelators like EDTA and EGTA can remove divalent cations responsible for maintaining cell function and integrity but their presence may inhibit certain enzymes, for instance, collagenase requires Ca(2+) for activity. Enzymatic and mechanical disaggregation is often a trial and error process to optimize the isolation of the epitope under investigation.

To study intracellular components e.g. 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 non-ionic 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 eventuality here, some standard protocols are given in this chapter.

EDTA Ethylenediaminetetraacetic acid
EGTA Ethyleneglycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid

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