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Principles of surface antigen analysis

This form of analysis is probably the most common but there are number of considerations to be made about cell preparation. This should be discussed with one of the contacts.

The key variables in any cytometric study are:

  • the nature of 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 which is satisfactory for one particular combination may not be suitable if applied to a different type of sample or if used to assay a different component.

If the sample already comprises cells in suspension, e.g. cultured cells or peripheral blood, then it may be possible to process and analyse it directly. However, if the cells to be studied occur within a solid tissue, they must first be dissociated and dispersed.

Flow cytometry usually requires ~0.5 ml of a cell suspension which ideally has a concentration in the range 10 5 cells ml -1 to 10 6 cells ml -1 . Cell suspensions should be free of tissue debris and large aggregates (clumps) of cells in order to avoid clogging the flow cell and tubing; if necessary, large particulates should be removed by filtering or centrifugating prior to analysis.

Dependent on the sample, the cells to be studied may be either, homogeneous, or heterogeneous, with respect to size and type. Flow cytometers can often distinguish different cell populations in heterogeneous samples solely on the basis of their forward and side light scattering characteristics. A fluorochrome-labelled antibody which is lineage or sub-set specific, (e.g. CD45 antibodies for leucocytes; CD3 antibodies for T-cells), or a mixture of fluorescent antibodies, can sometimes be used to identify the cells of interest in heterogeneous samples but this strategy decreases the number of fluorescence channels that can then be used for other purposes. Clearly, analysis is more efficient if the irrelevant cells can be removed from a sample. Thus, it is worth considering whether samples can be enriched for the required cells, either before or after labelling, without prejudicing the aims of the study.

When live cells are to be used, it needs to be decided how they should be maintained. Conversely, if fixed cells are to be used, it must be decided, how, and at what stage in the preparation procedure, they should be fixed.

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