Splenic foci technique

The splenic foci technique was developed independently in two laboratories (Kennedy et al., 1965 Playfair et al., 1965), with the same goal to obtain an assay for the precursors of antibody-forming cells. In principle, a small inoculum of donor spleen cells and antigen (red cells) is injected into lethally irradiated syngeneic mice. After 5 or more days the host spleens are cut into many slices, each of which is further cut into separate pieces. These are placed on petri dishes containing a layer of agar with embedded red cells. Antibody is allowed to diffuse out and complement-dependent lysis is observed around some pieces. In this way, it is possible to construct a three-dimensional map of foci of antibody-producing cells. 

In Table 2 there is presented a summary of estimates of precursor cells obtained using the splenic foci technique. Unfortunately, in most cases it is not possible to determine strictly whether T cells or B cells are limiting. Thus, it is not clear in many cases what precursor cell frequency has been calculated. Nevertheless, in cases where T cells are limiting, the frequency of B cells must be greater than values given in the table. 

Donor Antigen Recipient Transferred Time of Frequency Reference 

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Table 2. Frequencies of precursor cells estimated by splenic-foci technique...

Homing efficiency has already been discussed in the section on the splenic foci technique. The estimates for the fraction of specific cells which lodged in the spleen was in the range of 4-15% (see Chapter A). In experiments on transfer of an anti-DNP clone, Askonas et al. (1972) estimated the effective homing of B precursor cells in the spleen to be around 1 %. 

The estimates for precursor cells specific for red cell determinants were in the range of 0.2-3-3 X10 , for synthetic polypeptide antigens of limited heterogeneity O.3-3 X 10 , and for lipopolysaccharide 1 X10. These estimates are not corrected for the effective homing efficiency. For similar reasons to those mentioned in the section on the splenic foci technique, the above frequencies may be considered to be a minimum estimate. 

If microculture studies on the frequency of precursor cells are compared with those using the antigen-binding method, splenic-foci technique or in vivo dilution method, the following picture emerges ... 

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