The objectives in preparation and staining are obviously to obtain a single-cell suspension with quantitative fluorescence staining of the molecule(s) of interest. Easy to say, but not always easy to achieve. In some tissues and cell types this is straightforward but, in others it can be very difficult. Preparation and staining procedures in flow cytometry are legion and it would require the majority of this book to give a comprehensive account. Hence, only a brief review of the principles involved are summarized in this chapter, but details of some techniques are given in the relevant sections later on.

Disaggregation

Disaggregation is clearly the first step in the process of obtaining single-cell suspensions. Some tissues need little or no disaggregation and the best examples of these are cells of the heamopoetic and lymphoid systems. Other tissues, e.g. skin and some elements of the musculo-skeletal system, are extremely difficult to disaggregate effectively without cell damage. Generally, the ease with which disaggregation can be carried out is related to the function of the tissue. Epithelial cells which form a ‘barrier-layer’ at the environment interface have many types of intercellular connections, including desmosomes, which bind the cells of the epithelial surface tightly together. In contrast peripheral blood cells have no such connections. Two methods of disaggregation are available, namely mechanical and enzymatic, which can be, and often are, used in combination either with, or without, chelating agents.

Mechanical

Mechanical disaggregation procedures can only be really effective in very loosely bound structures such as bone marrow and lymphoid tissue (Shortman, 1972). The sheer forces due to syringing through fine-gauge hypodermic needles (Pretlow, Wein and Zettergen, 1975) are sufficient to separate bone marrow cells.