The FlowMetrixTM System is a multiplexed data acquisition and analysis platform for flow cytometric analysis of microsphere-based assays that performs simultaneous measurement of up to 64 different analytes. The system consists of 64 distinct sets of fluorescent microspheres and a standard benchtop flow cytometer interfaced with a personal computer containing a digital signal processing board and Windows95®-based software. Individual sets of microspheres can be modified with reactive components such as antigens, antibodies, or oligonucleotides, and then mixed to form a multiplexed assay set. The digital signal-processing hardware and Windows95-based software provide complete control of the flow cytometer and perform real-time data processing, allowing multiple independent reactions to be analyzed simultaneously. The system has been used to perform qualitative and quantitative immunoassays for multiple serum proteins in both capture and competitive inhibition assay formats. The system has also been used to perform DNA sequence analysis by multiplexed competitive hybridization with 16 different sequence-specific oligonucleotide probes.

It was recently hypothesized (1) that the two major immunoglobulin isotypes on the surface of B cells confer a different reactivity on the cell: IgM which appears early in ontogeny is a "tolerizing" receptor and IgD which appears later is a "triggering" receptor. As a first step in investigating this possibility, we have examined the relative susceptibility of neonatal B cells (which have virtually only IgM) and a population of adult B cells (which have both IgM and IgD) to tolerance induction. Materials and MethodsTolerance was induced in vitro using a variation of the technique described by Kettman (2) which is represented schematically in Fig. 1. Trinitrophenyl-human gamma globulin (TNP-HgG) was chosen as the tolerogen because of its well-characterized potential as an inducer of B-cell (and T-cell) tolerance in vitro (3). Spleen cells from adult (8-to 10-wk old) and neonatal (9-to 12-day old) (C57BL/6 × DBA/2)FI mice were harvested and treated with an antibrain 0-serum (4) and complement (C) to kill T cells. This treatment killed 46% of neonatal and 41% of adult spleen cells. The cells were washed and cultured at a density of 107 cells/ml in complete medium in the presence of varying concentrations of tolerogen (TNP17HgG). After an incubation period of 24 h, cells were washed three times with cold balanced salt solution (BSS) to remove the tolerogen and mixed with equal numbers of X-irradiated (1,500 R) spleen cells from adult mice which had been primed by intravenous injection of 200 tel of a 0.01% suspension of sheep erythrocytes (SRBC) 5-10 days before sacrifice (5). The addition of this cell population ensured that an excess of carrier-specific thymus-derived cells would be present during subsequent immunogen stimulation allowing measurement of functional bone marrow-derived anti-TNP and anti-SRBC precursor cells. The cell mixture was cultured at a cell density of 107 cells/ml in the presence of 100 tel of a 0.1% suspension of heavily substituted TNP-SRBC (6) per milliliter of culture. Cultures were incubated for 4 days (7) before being harvested and assayed (8, 9) for SRBC-and TNP-specific direct plaqueforming cells (PFC). In preliminary experiments, it was found that in the presence of excess helper cells, the anti-TNP and anti-SRBC PFC responses were linearly related to the number of "B" spleen cells cultured, irrespective of prior treatment of these cells with anti-O and C'. Similarly, treatment of the helper cell population with anti Ly-2.2 and C' [which has been shown to eliminate suppressor T cells (10, 11)] did not effect the ability of these cells to cooperate with the B-cell population nor did it effect the ultimate responsiveness of the B cells. In other control experiments, addition of a large excess of underivatized HgG (50 times the amount of TNP~THgG) during the phase of tolerance induction did not effect the dose response of the B cells to tolerogen. This result argues against the involvement of Fc receptors in tolerance induction using TNP-HgG.

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The in vitro primary response of mouse spleen cell suspensions to 2,4,6-trinitrophenyl(Tnp)-erythrocytes has been studied. The number of anti-Tnp

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