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When microcultures containing 2 x lo5 mouse spleen cells are stimulated with heterologous red cells and the response is measured by the production of plaque-forming cells or antibodies, some microcultures respond, some d o not. Microcultures which d o not respond t o a given antigen are still capable of responding t o another antigen. The response marks the presence of at least one precursor cell, probably a B cell. Most of the responding cultures would receive only one such precursor cell, and hence contain single clones of antibody-forming cells. The frequency of these precursor cells is calculated to be 5.5 x very small volumes (1 0 p1) are given in detail.Methods for handling and assaying large numbers of cultures in

Genome-scale microarray experiments for comparative analysis of gene expressions produce massive amounts of information. Traditional statistical approaches fail to achieve the required accuracy in sensitivity and specificity of the analysis. Since the problem can be resolved neither by increasing the number of replicates nor by manipulating thresholds, one needs a novel approach to the analysis. This article describes methods to improve the power of microarray analyses by defining internal standards to characterize features of the biological system being studied and the technological processes underlying the microarray experiments. Applying these methods, internal standards are identified and then the obtained parameters are used to define (i) genes that are distinct in their expression from background; (ii) genes that are differentially expressed; and finally (iii) genes that have similar dynamical behavior.

equine [ 161, bovine [ 171 or mule [ 151 and 5 % human serum [ 1 1 1 . Rat and human MLI contain 10 to 20 % fresh rat serum or human plasma, respectively [22, 231. Addition of reducing agents to these systems may result in lower serum requirements to achieve a better reaction, as suggested by the enhancement of nonspecific mitogen stimulation of human cells by L-cysteine [24].Studies of the capacity of homologous sera to enhance the mouse MLI revealed that sera from different strains of mice vary widely in stirnulatory capacity and that this capacity depended upon the combination of allogeneic spleen cells used. Two observations concerning stimulatory capacity of sera can be summarized: ( 1 ) the degree of enhancement is dependent upon the mouse strain from which the serum was prepared (C57BL > BALB/c > C3H) ; ( 2 ) serum autologous with the responder cells causes an optimal response at approximately 0.5 % serum. Some aspects of mouse serum described in the present report differ from those described for rat serum [ 2 2 ] . Unlike mouse serum, rat serum that was heat-inactivated or stored at -20 OC in the absence of a reducing agent was apparently unaffected as measured by its stimulatory capacity in the MLI. In addition, in contrast to mouse sera, sera of various rat strains did not show differential activity, but either possessed equal activity or were completely devoid of activity. It is unknown whether these differences are a consequence of different culture conditions or are inherent to the species.In summary, this mouse MLI possesses several advantages for in vitro studies of the immune response. The effects of serum and serum components on, and their possible relationship to cellular recognition of antigens can be investigated clearly without interference from heterologous sera. A dilution scheme for bone marrow-derived (B) cells has been applied to estimate the frequency of precursor cells specific t o sheep red cells (SRC) present in the spleens of nude mice. B cells were titrated over a range of 0.2 x lo5 -1.8 x l o 5 spleen cells of nude mice per microculture in the presence of a constant number of allogeneic spleen cells. Data from 6 limiting dilution experiments are presented. It is argued that only the B cells are being diluted out. The frequency of precursor cells specific for SRC was calculated by means of Poisson statistics. The estimates of these frequencies are in the range 0.9 x lo-' -2 x Correspondence: Jod Quintins, Basel Institute for Immunology, 487, Grenzacherstrasse, CH-4058 Basel, Switzerland Abbreviations: B cell: Bone marrow-derived cell PFC: Plaque-forming cell(s) SRC: Sheep red cell(s) T cell: Thymus-derived cell unequivocally that only one type of cellthe Precursor cellwas lacking in any nonresponding culture. A limiting dilution Em. J. Immunol. 1973.3: 392-397 Precursor cells in nude mice 393 The authors are indebted to N.K. Jerne for helpfur criticism during this work and to C.M. Steinberg for help in preparation of the manuscript. They also wish to thank J.R. Kettman for valuabl...