s

Sample Preparation Protocol

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 non-adherent 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 13).

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 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'-tetra acetic 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.

Preparation of Cells for Flow Cytometry

The most straightforward samples for flow cytometry are non-adherent cells from tissue cell culture. Here we describe methods for both tissue culture cell lines grown in suspension and adherent tissue culture cell lines. Analysis may be required of cells derived from other sources. We have protocols for the preparation of human peripheral blood mononuclear cells and for the preparation of peritoneal macrophages, bone marrow, thymus, and spleen cells.

It is recommended that all containers that have come into contact with human blood or cells should be considered hazardous waste and discarded appropriately.

Note: These methods provide general procedures that should always be used in conjunction with the product- and batch-specific information provided by the supplier.

A certain level of technical skill and immunology knowledge is required for the successful design and implementation of these techniques. These are guidelines only and may need to be adjusted for particular applications.