Introduction

When collecting and preparing samples for cytometric analysis, it is essential to use a protocol that enables the aims of the investigation to be fulfilled, because if samples are handled wrongly at the start, there is usually nothing that can be done later to remedy the situation. For many studies it may be possible to use, or to modify, an existing protocol; for others it may be necessary to devise a new one. In this chapter, the general principles relevant to collecting and preparing samples are discussed and some protocols described. Most were devised and validated using flow cytometry, but they should also be largely applicable to the newer technique of laser scanning cytometry.

Factors affecting the choice of preparation procedure

The key variables in any cytometric study are:

• the nature of the sample material and of the cells to be studied

• the cellular component(s) or molecule(s) that is(are) to be assayed

• the label(s)/probe(s) that is(are) to be used, and hence the signal(s) that is(are) to be detected.

All of these factors must be taken into account when considering how best to collect and prepare samples. A protocol that is satisfactory for one particular combination may not be suitable if applied to a diVerent type of sample or if used to assay a diVerent component.

The sample material and cells to be studied

Flow cytometers analyse the fluorescent and scattered light arising from cells illuminated in suspension, whereas laser scanning cytometers analyse the same parameters arising from cells illuminated on a microscope slide.