However, multiple antibody staining is more difficult, particularly, as is usually the case, if all the compensation antibodies are mouse monoclonals since the secondary antibody will bind to all the antibodies. The indirect method can be more sensitive several secondary antibodies may bind to a single primary molecule. When labelling cells in peripheral blood, the diluent often contains a detergent to lyse the erythrocytes. Washing is not always necessary the cells can be incubated with the antibodies in a small volume and diluted prior to measurement. Using the direct method, staining cells with several antibodies is straight forward. In the latter, the cells are incubated with the primary antibody, washed and then incubated with a secondary antibody carrying the fluorochrome ( Figure 5.1). In the former, the antibody is labelled with a fluorochrome and incubated with the cells in a single step staining procedure. Table 5.1. Some methods for permeabilising cells for intracytoplasmic stainingĬells can be stained either by a direct or an indirect method. Several manufacturers sell reagents which are mostly based on permeabilisation in detergent, usually saponin, and fixation in formaldehyde. Table 5.1 gives a list of some of the treatments that have been used successfully for different proteins. Many antigens are adversely affected by some fixatives and consequently the optimum procedure has to be determined for each protein under study. There is no standard procedure for staining intra-cellular antigens. Sometimes the cells are fixed after surface labelling, particularly if an intracellular antigen is also to be stained.įor cytoplasmic or nuclear antigens, the cells must be fixed and permeabilised to give the antibody access to the antigen. If surface antigens are to be stained, fresh unfixed cells are reacted with the labelled antibodies. In clinical samples, if nucleated cells are being studied, the red blood cells are usually lysed either by a brief exposure to distilled water, incubation with an ammonium chloride solution or with a weak detergent. Solid tissues are the most difficult but single cells can usually be obtained by a combination of mechanical stress, incubation with an enzyme or a combination of both. Lymphoid organs, such as thymus, will usually release single cells after mild mechanical treatment, for example, forcing the tissue through a coarse metal sieve of the type used as a tea strainer. The treatment might alter surface antigens. Cells grown adherent to a culture dish have to be removed from the dish, usually enzymatically. Cells from suspension cultures or from peripheral blood present few problems. The most important feature of sample preparation, as with all samples for flow cytometry, is the production of a suspension of single cells with few clumps and little debris.
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