Abstract: The hybridoma cell line B1-8delta 1 expresses an IgD antibody with specificity for the hapten 4-hydroxy-3-nitro-phenylacetyl (NP) Two monoclonal antibodies, each recognizing an idiotypic determinant (idiotope) in the variable region of antibody B1-8, and fluorescence-activated cell sorting were used for the isolation of spontaneous somatic cell variants whose antibody product lacks the expression of one of the two idiotopes The variant cells carry a mutation which is expressed in the variable region of the antibody heavy chains and which reduces the affinity of the antibody for hapten greater than 100-fold Idiotope loss variants of the selected phenotype are present in the B1-8delta 1 cell population at low frequency

Abstract: The expression of fibronectin by clones of 2102Ep, a human cell line derived from a testicular germ-cell tumor, was studied. In high-density cultures these cells retain an embryonal carcinoma phenotype and do not express the cell surface antigen SSEA-1. They do not synthesize fibronectin. However, when grown at low densities, many of the cells appear to differentiate, as indicated by a change in their morphology and their expression of SSEA-1. Such cultures also synthesize fibronectin. Further, when low-density cultures were fractionated into SSEA-1-positive and SSEA-1-negative subpopulations by fluorescence-activated cell sorting, fibronectin synthesis was confined to the SSEA-1-positive cells. The fibronectin produced by these cells exhibits an apparent molecular weight (261,000) slightly greater than that produced by diploid fibroblasts (250,000) and may represent an embryonic form. It is not laid down as an extracellular matrix and cannot be detected by immunofluorescence analysis of cell monolayers.

Abstract: A novel subset of human blood lymphocytes was isolated by means of labelling with monoclonal antibodies and fluorescence-activated cell sorting. In normal individuals, the new subset accounts for about 2% of the blood T lymphocytes. The cells of this subset bind monoclonal antibodies specific for T lymphocytes in general [e.g. OKT3, Hu-Lyt 3(9 . 6) and Leu-22] and they also form E rosettes. However, no binding is seen with monoclonal antibodies to T-lymphocyte subsets (OKT4, OKT8, Leu-2A and Leu-3A). Moreover, the lymphocytes of this new subset express neither Ia antigens nor membrane immunoglobulins. They do not bind OKM1, an antibody against cells of the myelomonocytic lineage that also reacts with natural killer cells, nor do they bind OKT6 or OKT10, specific for thymocyte antigens. The cells have a high specific gravity, a thymocyte-like pattern of lactate dehydrogenase isoenzymes and do not contain terminal deoxynucleotidyl transferase. Although these lymphocytes are viable, also after culture in vitro, and can be stored in liquid nitrogen, they are inert in all functional systems tested: they neither proliferate upon stimulation with mitogens or allogeneic cells, nor do they display suppressor or natural killer cell activity. A patient who was successfully reconstituted by bone marrow transplantation for severe combined immunodeficiency, was found to contain an abnormally high (25%--30%) fraction of these OKT3 positive, OKT4 and OKT8 negative cells among his circulating T lymphocytes.

Abstract: We have isolated by fluorescence-activated cell sorting an IgG1 expressing class switch variant (S24-1/47) from the IgG3-producing mouse hybridoma cell line S24/63/63. Both Ig bind the hapten 4-hydroxy-3-nitro-phenylacetyl (NP) with approximately the same affinity and fine specificity and are idiotypically indistinguishable. The apparent m.w. is 50,000 for the gamma 1 and 51,000 for the gamma 3 chain. The genomic DNA of IgG3-producing S24/63/63 cells contains a 14-kb Eco RI restriction fragment with C gamma 3 sequences representing a rearranged C gamma 3 gene. In the class switch variant S24-1/47 the C gamma 3 gene is deleted.

Abstract: Publisher Summary This chapter discusses the electronic cell sorting of hemopoietic progenitor cells. The hemopoietic system offers an exciting opportunity for the study of cell commitment, proliferation, and differentiation. Hemopoietic tissues are extremely complex, and hemopoietic cells form an interdependent regulatory network. This necessitates the purification of hemopoietic progenitor cells as well as their molecular regulators to study their interactions. The most primitive cell is a multipotential stem cell capable of extensive self-renewal as well as commitment to each hemopoietic cell lineage. The most commonly used assay for such cells is injection into lethally irradiated recipients where stem cells proliferate and form nodules or colonies in the spleen, hence the term colony-forming unit-spleen. Colony-forming cells, in turn, give rise to committed cells of lesser proliferative potential, termed cluster-forming cells, and finally to recognizable members of each hemopoietic lineage. The special power of fluorescence-activated cell sorting—the ability to assess quantitatively the level of binding of probe on a cell-by-cell basis—has been essential to attempts to purify the rare hemopoietic progenitor cells.

Abstract: Human natural killer (NK) cells separated initially by density centrifugation of lymphocytes (E+) forming rosettes with sheep red blood cells (SRBC), were further fractionated on gradients of bovine serum albumin (BSA). Low density fractions contained effector cells which displayed high cytotoxicity against the NK-sensitive erythroleukaemic cell line, K562. These low density cells, which expressed receptors for Fc and the monoclonal antibody OKMI, showed enhanced cytotoxicity when treated with lymphoblastoid interferon (IFN-alpha). They also showed an increased response to phytomitogen in comparison with unseparated cells or those recovered from high density fractions. Two lymphocyte subsets one of high and one of low lectin binding capacity were identified in the E+ populations by their reactivity with Lens culinaris agglutinin (LCA). High LCA binding was observed only in low density fractions and was associated with a marked enrichment of NK activity. This property was used to separate the NK active population in E+ cells by fluorescence-activated cell sorting (FACS). These data add a new dimension to the cell surface properties of human NK cells and suggest the presence of LCA-reactive glycoproteins which are either enriched in, or uniquely associated with, cells of the NK subset. The experiments indicate that lectins can serve as useful probes of lymphocyte function and provide the basis for effective cell sorting.

Abstract: Different genetic stains of avian RNA tumor virus (ATV) were labeled with the fluorescent membrane probe R-18 (rhodamine conjugated to a hydrocarbon chain) and cellular receptors for virus infection were analyzed on a rapid, single-cell basis by a multiparameter cell sorter. Chicken cells genetically susceptible to various R-18 ATV were found to adsorb much more virus, as measured by increased fluorescent binding, than did genetically resistant chicken cells. Virus binding to receptor sites could be saturated with increased concentrations of labeled virus. This binding could be altered by removal of the polycation, polybrene, indicating the important influence of electrostatic forces. Correlated time measurements of virus binding to single cells were taken with these fluorescence measurements allowing for a minute-to-minute study of the kinetics of viral adsorption to resistant and susceptible cells. The ratio of fluorescence (proportional to the number of virions bound per cell) to light scatter (proportional to cell surface area) on a cell-to-cell basis was analyzed to examine the heterogeneity in fluorescent virion bound per unit cell surface area within a given cell type. With these calculations, it was found that a large amount, but not all, of observed fluorescence heterogeneity merely reflects differences in cell surface areas. However, there are significant differences in viral receptor site densities within this supposedly homogeneous population of cells. This study represents a successful application of fluorescent membrane probes and flow cytometry to the study of cellular responses to viral infection at the single-cell level. Sine large numbers of cells can be examined rapidly, small subpopulations of live virally susceptible or resistant cells can be cloned by multiparameter cell sorting.

Abstract: The relationship between cell surface antigen expression and function in cytotoxic T cells directed against a non-H-2-restricted plasmacytoma antigen was examined using Lyt-2- variants of a continuous T cell line. This cytotoxic cell line originally had expressed Lyt-2 on all the cells as detected by flow microfluorometry, but after several months of culture, Lyt-2- variants spontaneously developed. Using cell sorting and limiting dilution cloning, Lyt-2- and Lyt-2+ cell lines were obtained, cultured and analyzed. These analyses indicated that such cytotoxic cells had similar activity regardless of Lyt-2 phenotype. This observation supports the concept that the Lyt-2 molecule is not obligatory for recognition and killing by cytotoxic T cells and emphasizes the need to clarify the precise relationship between cell surface phenotype and T cell function.

Abstract: The effect of 5 mg hydroxyurea (HU) i.p. on epidermal DNA synthesis in female hairless mice was assessed by measuring labelling indices and specific activity after 3HTdR injection, flow cytometry (FCM) and cell sorting of prelabelled basal cells. HU causes an almost immediate block in DNA synthesis lasting until 2-2.5 h. During this time the fraction of cells in S remains stationary, 1.20 of normal. From 2.5 to 12.5 h DNA synthesis is resumed, but in cells recruited from G1 or G0. The HU-blocked cells do not move out of S until after 12.5 h. Hence, from 2.5 to 12.5 h, the fraction of cells in S increases to 2.5 of normal, which means that entry into S is open, but exit is blocked. From 12.5 h flux through S is high. The blocked cells are now released and the fraction of cells in S falls to 0.7 of normal at 24.5 h. At 36.5 h a probable new wave of DNA synthesis is indicated. The results also show that 3HTdR is available for at least 20 min after i.p. injection. The consequences of these results for the interpretation of the effect of HU pretreatment on methylnitrosourea skin carcinogenesis are discussed.

Abstract: Macrophages represent a functionally heterogeneous group of cells which belong to the mononuclear phagocyte system (1). Heterogeneity may exist between macrophages from different organs as well as among macrophages within one organ (2,3). Heterogeneity has been defined by differences of Ia-antigen expression (4,3); monoclonal antibodies (5,6) against cell surface determinants; receptors for the C3 complement component or the Fc part of IgG molecules; cell size (7,3); enzyme activities e.g. phosphatase, nucleotidase (8) peroxidase (9) and transglutaminase (10); wheat germ lectin binding (11); tumor cytoxicity (12); or phagocytosis (13) and adherence. Classification according to several parameters is necessary to identify small subpopulations of macrophages (1). Flow cytometry is a particularly useful method for this purpose, especially because functional parameters of living cells can be measured simultaneously at the single cell level in a fast and accurate way. Such parameters include cytoplasmic (14,15) or lysosomal (16) enzyme activities, transmembrane potential (17,18), intracellular pH (19) and phagocytosis. The use of vital stains also permits cell sorting. Sorted cells can be recultivated and further analyzed. Macrophages are often characterized as cells with high esterase activity (20,21,22), although there are some reports on low esterase activity in macrophages (23,24,25). It was the purpose of this study to characterize the low activity macrophages in more detail.

Abstract: We have attempted to purify human bone marrow granulocyte-macrophage progenitor cells utilizing purified stimulating factor, indirect immunofluorescence and cell sorting. These cells can be labelled by sequential incubation with colony-stimulating factor (CSF), rabbit anti-CSF antibody and fluorescently labelled goat anti-rabbit gamma globulin. Flow photometry demonstrated that binding of CSF to marrow cells varied with CSF concentration and duration of incubation. Marrow cells were pre-concentrated by incubation with carbonyl iron followed by Percoll gradient centrifugation and then labelled by this indirect immunofluorescence method. Sorting of these cells yielded up to 95-fold purification of colony-forming cells and fractions containing in excess of 10% colony- and cluster-forming cells.

Abstract: Publisher Summary This chapter focuses on size distribution and β2-microglobulin content of human natural killer (NK) cells analyzed by fluorescence-activated cell sorting (FACS). In a study described in the chapter, normal peripheral blood mononuclear cells were isolated by Isopaque–Ficoll method and stained with a FITC-conjugated rabbit anti-β2-M antiserum. The cells were subsequently processed on a FACS II machine. In the chapter, the dot plot screen of the FACS II from a typical experiment is shown, each cell being represented by a dot giving its size (or forward light scatter, x-axis) and the fluorescence intensity (y-axis). Both lymphocytes and monocytes—as verified by Giemsa-stained smears—can be delineated and lymphocytes can be further subdivided into four lymphocyte subsets according to their β2-M content and size. The FACS was set up to sort the 20% least and the 20% most fluorescing cells, and it was found that cells with low β2-M content, irrespective of size, always exhibited lower NK than the high β2-M containing cells and that IFN-inducible NK cells moreover were found in the β2-M rich cell fractions, indicating that the association between β2-M and NK might be stronger than that between NK and cell size.

Abstract: In continuation of a previous study, the quantitative fine structural characteristics of the average normal human pan-T lymphocyte and its subsets in the peripheral blood has been established using stereological methods. T cell subpopulations were isolated and identified by means of monoclonal T3, T4 and T8 antibodies and a fluorescence-activated cell sorting machine. Comparative studies on ultrastructural morphology were made between the E-rosetting lymphocyte and the T3-positive cell, both markers of the pan-T cell, and between the functionally different T4- and T8-reactive subsets, defining the helper/inducer and suppressor/cytotoxic T cell subpopulation respectively. No significant differences were recorded between the E-rosetting and the T3+ lymphocyte except for minor deviations regarding the surface of the plasma membrane. In comparison with the T4+ lymphocyte, the T8+ cell showed a larger cell volume and cell surface area and decreased nucleo/cytoplasmic volume and surface ratios. The increase in cell size was the result of greater volumes of residual cytoplasm as well as of intracytoplasmic organelles, such as mitochondria, RER, Golgi apparatus and granules, whereas nuclear parameter estimates were concordant. The structural deviations between the T4+ and the T8+ subsets are discussed in the context of their different functional capacity as helper and suppressor/cytotoxic cells.