Although it is well accepted that immunocornpetent T lymphocytes can be derived from blood-borne stem cells by a series of developmental events that occur within the mouse thymus (1, 2), the maturation sequence, inductive signals, and precursor-product or lineage relationships involved in this process are as yet poorly understood. An extensive literature suggests that cortical-type thymocytes are the precursors of the medullary-type thymocytes. ~ This conclusion is based on early data obtained from in vivo labeling experiments, as well as in vitro functional assays using isolated subpopulations of thymocytes, either in the presence or absence of added factors (for review, see Refs. 4-7). This concept has, however, been challenged by other in vivo labeling studies (8, 9), and more recently by in vitro functional assays using highly purified subpopulations, and by limiting-dilution analyses, leaving open the issue of the actual relationship between these two categories of thymocytes (10-12).Several approaches have been taken to identify the intrathymic precursors to cortical and/or medullary-type thymocytes. The results of several studies on fetal thymic development in situ or in vitro organ cultures (12-21), or chimeras prepared with fetal thymus grafts (21), suggest that cells with cortical and medullary phenotypes are derived from early Thy-1 +, Ly-l-low, Lyt-2-, L3T4-thymocytes. In the most direct demonstration of a precursor-product relationship, Ceredig, et al. (19) showed that a proportion of purified Lyt-2-fetal thymocytes will convert to Lyt-2 + after short-term in vitro culture. In addition, mature functional T cells (cytolytic and interleukin 2-producing) can be derived from organ cultures made from early fetal thymuses, which contain only Thy-1 +, Lyt-2-, L3T4-thymocytes, in the absence of any further influx of cells (16, 2O).Recently, we reported (21-24) a minor subpopulation of adult mouse thymocytes (<5%) that expresses low levels of Ly-1, and no Lyt-2 or L3T4 surface 3"he terms cortical-type and medullary-type are used here to imply a surface phenotype (Lyt-2 +, L3T4 + for cortical, and Lyt-2 +, L3T4-or Lyt-2-, L3T4 + for medullary, respectively) rather than an anatomical location. However, cells with these major phenotypes have been localized to the cortex and medulla in histological sections (3).

The accurate measurement of ionized intracellular calcium [Ca++] Full understanding of lymphocyte transmembrane signaling requires detailed analysis of the rapid response to occupancy and/or crosslinking of membrane receptors for antigen, lymphokines and hormones as well as identification of the receptor molecules that mediate these processes. Heterogeneity of cell phenotype and functional responses, as well as clonal distribution of antigen receptors, requires analysis at the single cell level of the intracellular second messengers produced by these interactions. Much recent evidence indicates that the level of cytoplasmic free ionized calcium [Ca++]i is a critical second messenger (1) in the activation of both T (11,23,28) and B (2,3,4,12,16,19) [Ca++]i determination by flow cytometry offers several experimental advantages. It provides fully objective, highly quantitative measurements. The technique is applicable to any type of dissociated cells. Cross-correlation with other physiologic parameters and surface antigens can be obtained. Preparative sorting for functional correlations and selection of mutants to dissect pathways are both feasible.The newly available fluorescent chelator, indo-1 (9), has the properties required for single cell calcium measurement in the flow cytometer. When excited near the optimal wavelength of 356 nm, the fluorescence emission maximum shifts from 485 nm for free dye to 404 nm for the Ca++/indo-l complex with minimal change in fluorescence intensity. The ratio of fluorescence emission at these wavelengths reflects the fraction of Ca++-bound dye molecules and thus provides a quite precise measurement of [Ca++]i that is independent of the individual cell's dye content. Such ratio determinations eliminate variability due to heterogeneity in dye uptake and cell size.

A method to preserve stained human lymphocytes for subsequent cell surface analysis by flow cytometry (FCM) is described. Cells stained with fluorescein isothiocyanate (FITC) and phycoerythrin (€'E)-conjugated monoclonal antibodies and then fixed in 1% paraformaldehyde, followed by extensive washing and resuspension in 1% BSA medium, could be stored at 4°C for at least 2 weeks prior to FCM analysis without significant alteration in the light scatter or fluorescence properties of the cells. Furthermore, the method was also suitable for analyzing lymphocytes that express T-cell activation markers in certain disease conditions. In addition, we have identified monoclonal antibody combinations that discriminate different lymphocyte subsets that are satisfactory for multiparameter analysis after 2 weeks of storage. This method should prove useful for enumerating lymphocyte subsets in field study sites remote from flow cytometry laboratories.Key terms: Paraformaldehyde, filariasis, systemic lupus erythematosusThe lymphocyte population has been shown to be heterogeneous with respect to cell surface antigens (21). The functional properties of cells correlate strongly with these lineage-specific differentiation antigens (14). The subpopulations of B cells (1) and T cells (10) can be quantitated by flow cytometry (FCM) based on the relative expression of two or more cell-surface markers (8), and this approach has been used to define abnormal lymphocyte subsets in a variety of clinical conditions (9, 11).For such clinical studies, fresh lymphocytes (7) have generally had t o be used, since conflicting results have sometimes been observed with cryopreserved cells (31, an observation suggesting that certain differentiation antigens may be cryolabile. Thus, in most studies, cells are stained by conventional immunofluorescence techniques and analyzed either fresh or shortly after paraformaldehyde fixation (13). For studies carried out at sites remote from a flow cytometer, this protocol, however, creates practical problems, since the effect of longterm fixation on FCM analyses has not been well defined.Therefore the purpose of this study was to develop the optimal method of preserving stained cells for as long a time as possible. The basic requirement of such a technique would be that it permit 2-3 weeks storage of the cells prior to analysis without significant alteration of the light scatter and fluorescence properties of the cells. In this report, we describe such a method and define monoclonal antibody reagents satisfactory for studies requiring prolonged storage of FCM samples prior to analysis. MATERIALS AND METHODS Study PopulationThirteen individuals were studied. Eight were healthy North Americans, two were normal volunteers from New Delhi, India, two were patients with systemic lupus erythematosus, and one was an Indian patient with tropical pulmonary eosinophilia.Preparation of Fixative A 1.0% (w/v) solution of paraformaldehyde (Eastman Kodak Co., Rochester, NY) was prepared by dissolving paraformaldehyde in phosphat...

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