Abstract: The question of whether the initial regulatory event, which directs an uncommitted precursor cell toward terminal differentiation, is cell cycle phase specific was examined using the human promyelocytic leukemia cell line, HL-60 While the HL-60 system does not reflect all of the features of normal hematopoiesis, it does provide a relatively well-defined in vitro experimental system which can be useful for examining aspects of the differentiation process HL-60 cells were induced to undergo myeloid differentiation by retinoic acid The subsequent differentiation kinetics of HL-60 populations initially enriched in different cell cycle phases was measured This was compared to the cellular uptake of retinoic acid as a function of cell cycle position If the initial differentiation-regulating event were cell cycle phase independent, then the kinetics of differentiation would be independent of the cell cycle status of the initial population Flow cytometric cell sorting, based on cellular narrow angle and orthogonal light scatter intensity spectra, was used to select G1-enriched and S + G2 + M-enriched cell populations without pharmacological perturbation These two populations were each induced to undergo myeloid differentiation with 10(-6) M beta-all-trans-retinoic acid The kinetics of G1/0 arrest associated with terminal cell differentiation, as well as phenotypic differentiation, assayed by development of oxidative metabolism, was measured for both populations The kinetics of differentiation differed for the two populations, indicating that the initial differentiation-regulating event was cell cycle phase specific For both of the initial cell populations, significant phenotypic differentiation followed approximately 24 hr after enrichment in the relative number of S-phase cells When exponentially proliferating HL-60 cells were exposed to a 1-hr pulse of 10(-5) M [3H]retinoic acid and then flow cytometrically sorted by DNA content, cells in late S + G2 + M had an approximately 10-fold higher uptake than cells in G1 or early S The results indicate that cellular regulation of myeloid differentiation first becomes responsive to the inducer, retinoic acid, in S phase when uptake is enhanced

Abstract: Treatment of Daudi cells with human lymphoblastoid interferons for up to 5 days progressively inhibits cell proliferation. For the first 3 days cells continue to grow but with prolonged doubling times; subsequently, net proliferation ceases and is accompanied by a loss of cell viability. We have investigated the changes in labelling of DNA with radioactive precursors which occur during the first phase of the response to interferon treatment. We have shown previously [Gewert et al. (1981) Eur. J. Biochem. 116, 487-492] that inhibition of incorporation of [3H]thymidine into DNA can be accounted for by impairment of thymidine transport and thymidine kinase activity. In spite of this inhibition, the total intracellular dTTP pool is larger in interferon-treated than in control cells. Because of these changes it has been necessary to use other methods to determine whether interferon treatment inhibits the overall rate of DNA synthesis. The results of experiments employing (a) moderately high thymidine concentrations or (b) incorporation of radioactivity from deoxynucleoside triphosphates into DNA in detergent-lysed or permeabilised cell systems indicate that there is in fact relatively little inhibition of the overall rate of DNA synthesis in cells exposed to up to 100 units/ml of interferons for at least 48 h. Furthermore, a similar proportion of cells incorporate [3H]thymidine in control and interferon-treated cultures and there is only a small decrease in the number of cells in S phase after interferon treatment, as revealed by fluorescence-activated cell sorting. These results indicate that cell proliferation may be regulated in this system by a mechanism in which there is a loss of coordination between the initiation of DNA synthesis and the subsequent events required for cell division.

Abstract: A monoclonal antibody, anti-Leu 11a (NPK-15), was generated against human large granular lymphocytes (LGL). Anti-Leu 11a reacted with the majority of Percoll gradient-enriched LGL cells, a subpopulation of peripheral blood lymphocytes (PBL) approx (approximately 10-20%), and most granulocytes, but not with a significant number of monocytes, T lymphocytes, or erythrocytes. Cell sorting experiments demonstrated that the Leu 11a+ population encompassed essentially all functional natural killer (NK) cells in the peripheral blood. Two-color flow cytometry analysis of PBL populations stained with anti-Leu 11a and anti-Leu 7 revealed the existence of four distinct populations: Leu 11a-, 7+; Leu 11a+, 7-; Leu 11a+, 7+; and Leu 11a-, 7-. The Leu 11a+ population did not appear to include cells marked with the T cell-associated antigens Leu 1, Leu 2, or Leu 3. The existence of a cell surface antigen common to granulocytes and NK cells, which is capable of distinguishing subpopulations of Leu 7+ cells, provides a useful probe to analyze the nature of the NK lineage.

Abstract: Selected cell synchrony techniques, as applied to asynchronous populations of Chinese hamster ovary (CHO) cells, have been compared Aliquots from the same culture of exponentially growing cells were synchronized using mitotic selection, mitotic selection and hydroxyurea block, centrifugal elutriation, or an EPICS V cell sorter Sorting of cells was achieved after staining cells with Hoechst 33258 After synchronization by the various methods the relative distribution of cells in G1, S, or G2 + M phases of the cell cycle was determined by flow cytometry Fractions of synchronized cells obtained from each method were replated and allowed to progress through a second cell cycle Mitotic selection gave rise to relatively pure and unperturbed early G1 phase cells While cell synchrony rapidly dispersed with time, cells progressed through the cell cycle in 12 hr Sorting with the EPICS V on the modal G1 peak yielded a relatively pure but heterogeneous G1 population (ie early to late G1) Again, synchrony dispersed with time, but cell-cycle progression required 14 hr With centrifugal elutriation, several different cell populations synchronized throughout the cell cycle could be rapidly obtained with a purity comparable to mitotic selection and cell sorting It was concluded that, either alone or in combination with blocking agents such as hydroxyurea, elutriation and mitotic selection were both excellent methods for synchronizing CHO cells Cell sorting exhibited limitations in sample size and time required for synchronizing CHO cells Its major advantage would be its ability to isolate cell populations unique with respect to selected cellular parameters

Abstract: Antisera with specific reactivity towards adipocyte cell surfaces were prepared and characterized. These preparations, absorbed to remove reactivities toward other cell types were used in an indirect labelled second antibody cellular immunoassay which distinguished bovine adipocyte precursors from fibroblasts and which allowed the progress of precursor cell differentiation in culture to be monitored with precision and sensitivity. The assay could be modified to use fluorescent, rather than radioactively-labelled, second antibody preparations and the changing reactivity of differentiating precursors to be visualized. Labelling of preparations in this way also allowed precursor cell populations to be analysed and quantitated using fluorescence-activated cell sorting technology.

Abstract: Monocyte/macrophage-mediated cytotoxicity requires the generation of activated oxygen radicals, which can be measured by chemiluminescence (CL). To investigate whether natural killer (NK) cell activity required activated oxygen species, both cytotoxicity against K562 target cells and CL were measured in cell populations of human peripheral blood. The following results were obtained: (a) Peripheral blood mononuclear cells (MNC) showed NK activity and a response in CL, which could be induced by viable or paraformaldehyde-fixed K562 target cells as well as by latex particles, (b) Both T cells and non-T cells exhibited NK activity, but T cells gave no K562- or latex-induced CL responses, (c) Depletion of phagocytic cells from MNC abolished CL, but only marginally affected NK activity, (d) Reconstitution of phagocyte-depleted MNC with adherent cells revealed a superadditive enhanced CL response, but had no augmenting effect on NK activity, (e) Phagocyte-depleted cell populations, enriched for NK activity by density gradient centrifugation, did not respond in K562- and latex-induced CL. (f) MNC, highly enriched for NK activity by cell sorting with a cytofluorograf using the fluorescein isothiocyanate-labeled monoclonal antibody anti-Leu-11a, responded only with reduced CL, whereas the NK activity was enriched up to 45-fold. From these results it is concluded that NK cell-mediated cytolysis of K562 target cells and K562-induced CL are not functionally correlated, but represent properties of two distinct cell populations, namely NK cells and monocytes.

Abstract: With the advent of monoclonal technology, it has been possible to define surface membrane markers which discriminate among subpopulations of lymphoid and myeloid cells that relate to both ontogeny and functional heterogeneity. With reference to the myelomonocytic lineage, the efforts of several laboratories have resulted in the development of monoclonal reagents that distinguish antigens shared by myeloid cells and cells of other lineages (lymphoid and erythroid), determinants specific for the myeloid series (in some cases including the monocytic-granulocytic stem cell), and markers selective for fully differentiated monocytes and macrophages. These monoclonal reagents have therefore made it possible to associate membrane structural features with morphologically identifiable stages of normal myeloid differentiation, and with neoplastic cells that represent a malignant proliferation of a given stage of maturation. Moreover, antibodies specific for monocytes and macrophages have provided methods (e. g., complement lysis, rosetting, cell sorting) for either depleting or purifying these cells in order to investigate the function of monocytes in immune phenomena.

Abstract: A new method for measuring cell survival at low doses of ionizing radiation has been developed through the use of flow cytometric cell sorting on the basis of Coulter volume signals. The cell sorter is capable of deflecting a precisely known number of cells directly into culture dishes, thus eliminating any errors associated with cell dilution and volume sampling. The use of Coulter volume signals as the sorting parameter is shown to be noncytotoxic for a variety of cell lines. Comparison of radiation survival curves measured above the 10% survival level by either the cell sorter or standard dilution assay demonstrates the increased precision of the cell sorter technique. Because of these advantages of cell sorting over conventional methods of plating cells, this technique has many applications in the field of radiation biology and other studies of cell survival.

Abstract: The stability of expression of a membrane antigen on human FME melanoma cells was investigated by means of flow cytometric cell sorting and analysis. The melanoma-associated 250 kd antigen was strongly expressed on all cells, as recognized by binding of the monoclonal antibody 9.2.27. By flow cytometric cell sorting, cells of high and low antigen expression were isolated, and the difference in antigen expression between the two populations was examined as a function of time in culture. Immediately after sorting, the median fluorescence intensities of the two populations differed by a factor of 2.7. After the first few days in culture, much of the range in antigen expression of the parent population was regenerated. However, a lasting difference in antigen expression was established, corresponding to 50% higher density of antigen on the cells sorted for high fluorescence intensity, compared to those sorted for low intensity. After trypsin treatment, which removed the antigen from the cell surface, normal antigen expression was regained after 2-3 days in culture, with the same difference between the two populations as before the trypsin treatment. The stability of the established difference in antigen expression between the two sorted subpopulations indicates that expression of this antigen is a precisely controlled, heritable characteristic of the FME melanoma cells.

Abstract: We have transferred the gene coding for the Thy-1.2 alloantigen into a Thy-1.1-bearing T-cell lymphoma. BALB/c thymocyte DNA, precipitated with calcium phosphate, was used to effect the transfer. We report the stable transformation of lymphoid cells by total cellular DNA. To our knowledge, this has not been previously reported. Transient expression of the transfected gene could be detected by flow cytofluorometry, and 5% (range, 1.5%-11%) of the recipient cells had Thy-1.2 antigen detectable on their surface 48 hr after transfer. "Stable" transformants were isolated by repeatedly selecting for cells expressing the Thy-1.2 antigen, by use of fluorescence-activated cell sorting or "panning." No metabolic selection was required. The transferred gene, detected by Southern blotting, encoded a product that is indistinguishable from the normal antigen by immunoprecipitation and NaDod-SO4/PAGE.

Abstract: When added to a mixed lymphocyte culture, cells in T cell colonies grown from bone marrow (BM) suppressed the development of cytotoxic activity against H-2 antigens shared by the colony cells and the stimulator cells, apparently by inactivating cytotoxic T lymphocyte precursor cells (CTLP). From the point of view of the added suppressor cells, the suppression was against self-reactive cells. The suppressor cells were resistant to gamma irradiation (1500 rds) but sensitive to UV irradiation. Inactivated CTLP separated from the suppressor cells by cell sorting could not be reactivated on being recultured with fresh stimulator cells, suggesting the suppression is irreversible. There was a critical time window, extending roughly from 20 to 40 h after culture initiation, during which the suppressor cell had to be present if CTLP were to be inactivated. During the first 20 h and after 40 h of exposure to stimulator cells, CTLP were resistant to the suppressor cell. Direct experimental evidence is presented against the possibility that the suppressor cells derived from BM colonies act by augmenting the production of Lyt-2+ suppressor cells from the responder population which then produce the suppression, or that the suppressor cells interfere with an early interaction between CTLP and stimulator cells. We conclude that the suppressor cells in T cell colonies grown from BM act directly on activated CTLP and permanently inactivate them.

Abstract: Publisher Summary This chapter discusses the development of techniques for the isolation and separation of bone cell populations. One line of differentiation is to the preosteoblast, a glycogen-rich, distinctly identifiable cell, which is the immediate precursor of the osteoblast. The osteoblast is a mature nonproliferating end-stage cell, which synthesizes and secretes the components of new bone matrix and participates by as yet undefined mechanisms in the mineralization of that matrix to form bone. The two cell types, namely, the preosteoblast and osteoblast, are the primary targets of efforts to isolate cells for the study of bone formation in vitro. The classic view of an osteoclast is a multinucleated giant cell anchored to a surface of bone undergoing active resorption. The morphology of this cell with its specialized ruffled border and surrounding clear zone has been extensively characterized. The methods that have been applied to bone cell separation include differential surface adhesion, unit gravity sedimentation, filtration, isopycnic centrifugation, fluorescence-activated cell sorting, and free flow electrophoresis (FFE). Osteoblastic and osteoclastic cells can also be separated by FFE. This method of cell separation has not gained wide acceptance; the apparatus that is required is expensive and is not commonly found.

Abstract: We present a method of synchronizing cells in G1-, S-, and G2M-phases employing sequential centrifugal elutriation and viable flow cytometric cell sorting of Hoechst-33342 stained Chinese hamster ovary cells. G1- and S-phase cells can be separated to greater than 99% homogeneity and G2-M to 70% purity. Most of the 30% contamination in the G2-M fraction was due to S-phase cells, whose reproductive integrity could be eliminated through the use of high specific activity 3H-TdR. There were minimal toxic effects or perturbations to growth following the selection procedures. The most significant limitation of this technique appears to be the rate of cell sorting, which, with current equipment, is approximately 3,000 cells per second.

Abstract: The sorting behavior of mixtures ofD. discoideum cells which had been developed for different lengths of time was examined. Cells developed for 4 and 8 h were mixed together and allowed to form slugs. Within the slugs, 8 h cells sorted to the anterior prestalk region while 4 h cells sorted to the posterior prespore region. These results indicate that the more developed a cell is, the more likely it is to become part of the prestalk zone in the slug. They are also consistent with the differential adhesion and chemotaxis hypotheses as a mechanism for cell sorting since cells become more adhesive and chemotactically responsive as development proceeds.

Abstract: Environmentally induced mutations, especially those involving large scale genetic damage such as deletions and chromosome loss, are of central importance in the production of human genetic disease and cancer. We have developed a methodology, the AL assay, that permits detection of such extensive genetic changes which often escape detection in other systems in which they are lethal. The AL assay employs a human-Chinese hamster ovary cell hybrid that retains a single human chromosome, number 11. A set of specific cell surface antigens result from genes located on opposite arms of this chromosome. Exposure to mutagens produces mutants which form colonies in the presence of complement and specific antiserum that kill nonmutant cells. The frequency and pattern of marker loss provides a measure of single gene mutation, large and small deletion, and loss of the entire chromosome 11. We have employed the indirect fluorescein conjugated isothiocyanate (FITC) technique and fluorescence-activated cell sorting (FACS) to remove spontaneous mutants from the initial population. The 100-fold reduction in background thus far achieved should allow accurate analysis of mutation by ionizing radiation at doses of less than 10 rad.

Abstract: Human low-molecular-weight (2', 5')A synthetase is induced in certain human X mouse somatic hybrid cell lines when these cells are treated with mouse interferon. We have assigned the gene coding for this interferon-inducible antiviral enzyme to human chromosome 11 by somatic cell genetic techniques (1). Fluorescence-activated cell sorting for cells expressing or lacking 4F2 antigen in two independently derived, chromosome 11-containing hybrid cell lines separated the cells into subpopulations of cells that had retained or segregated chromosome 11, respectively (2). We used these subpopulations to confirm our gene assignment by demonstrating that retention of chromosome 11 was required for expression of human (2', 5')A synthetase.

Abstract: Publisher Summary This chapter discusses the cell labeling and separation using immunospecific microspheres. The separation of a complex mixture of cells into specific cell populations is fundamental for analysis of the molecular properties, structure, and function of these cells in normal physiological processes and in pathological conditions. The basic ingredients include cells bearing defined antigens or receptors, ligands that bind with a high degree of selectivity and affinity to these cell surface sites, the microspheres that can interact with the specific ligand, and the separation method based on the characteristic properties of the microspheres. The first involves the labeling of cells displaying the target receptor or antigen with antibodies or other ligands directly or indirectly bound to the microspheres. The methodology is identical to the immunofluorescent and immunoelectron microscopic techniques developed for the identification and quantification of specific cells. The second step involves the separation of labeled cells from unlabeled cells. Any of a variety of separation techniques that discriminate microsphere-labeled cells from unlabeled can be used. Such techniques include centrifugation based on the equilibrium density or sedimentation velocity, electrophoresis based on the surface charge, magnetophoresis or magnetic chromatography based on the magnetic properties, and fluorescence-activated cell sorting based on the fluorescent properties of the microsphere-labeled cells. These separation techniques require that cells be present in suspension and be free of aggregates.

Abstract: Cell sorting is a way to isolate viable subpopulations of cells present in a mixture. The drawback of the isolation method is the shortage of material for subsequent biochemical determinations. We have employed a combination of (ultra-) microchemistry and cell sorting to overcome this problem. The methods enable determinations of protein and several enzyme activities on triton extracts of 5,000–10,000 sorted cells. In addition, using ultramicromethods we could determine enzyme activity in single sorted cells. This combination of methods is used for clinical genetic studies on heterozygote detection in Fabry's disease, an X-linked genetic disease. More-over, microchemistry is used to study enzyme activities in sorted autofluorescent “aged” fibroblasts.

Abstract: Most cells in the normal adult mouse thymus express Thy-1 glycoprotein but do not express Pgp-1 glycoprotein. In contrast, cells of the mouse B-cell lineage are Thy-1 negative and Pgp-1 positive. Somatic cell hybrids between pseudodiploid Thy-1+, Pgp-1− T-cell lymphomas and pseudotetraploid Thy-1−, Pgp-1+ Abelson-leukemia-virus-induced cell lines do not express detectable cell-surface Thy-1 but show activation of the T-cell Pgp-1 glycoprotein. Hybrids between pseudodiploid lines, in contrast, show extinction of Pgp-1. Thy-1+ or Pgp-1+ revertants were isolated by cell sorting from hybrids in which extinction occurred, demonstrating that all genes required for expression of these cell-surface antigens were present in antigen-negative hybrids. Thy-1− hybrids did not contain detectable cytoplasmic Thy-1 messenger RNA, while Thy-1 message could be detected in parental lines and Thy-1+ revertants. No obvious rearrangements of the Thy-1 structural genes could be demonstrated in Thy-1− hybrids and their Thy-1+ revertants, nor could rearrangements be demonstrated when parental cells and Thy-1− hybrids were compared. These results are consistent with the idea that diffusible gene products regulate both Thy-1 and Pgp-1 expression in these hybrids. These products act in a gene dosage-dependent manner in somatic cell hybrids. Regulation of Thy-1 is at the level of either messenger RNA transcription or processing.

Abstract: The relationship between gene expression in a cell and the protein patterns that can be obtained by two-dimensional polyacrylamide gel electrophoresis has been widely discussed in recent years. Evidence to support the validity of a strong connection between the interworkings of the cellular protein synthesis mechanisms, their relationship to gene expression, and the unique patterns obtained from the gels has been published.’-’ Thus, we believe that the high-resolution gel technique described by O’Farrell in 1 9754 can be used to search for changes that occur in relation to a specific disease and perhaps ultimately contribute to a better understanding of the disease process itself. It has been possible for us to establish a normal pattern of protein expression for human leukocytes by two-dimensional gel analysis of Ficoll-paque purified cells from the blood of hundreds of different individuals. These gels are highly reproducible in the position and density of leukocyte spots on patterns from one individual to another, the notable exceptions being polymorphic proteins, such as the HLA antigens. Dissection of human leukocytes into specific cellular subsets by the use of monoclonal antibodies and fluorescence-activated cell sorting allowed us to establish marker proteins for each individual subset of cells.’ Based on this knowledge, it is possible to differentiate changes in the leukocyte gel patterns due to subpopulation redistribution and those due to apparently abnormal protein expression. Traditionally, abnormal leukocytes involved in a variety of hematologic disorders have been distinguished from their normal counterparts by morphologic, histochemical, and cytogenetic differences. In recent years, immunologic techniques have been applied in hematologic analysis and have improved our understanding and classification of immune disorders. Preliminary two-dimensional gel analyses of abnormal leukocytes from patients with leukemia, infectious mononucleosis, and rheumatoid arthritis have been shown to be capable of detecting reproducible abnormalities in these patterns, which are characteristic of the specific disease.”’ Further analysis of proteins that appear to be abnormally regulated or expressed in hematologic diseases may lead to the discovery of differences that could be attributed to the cause of the disease, the presence of existing disease, or the suggestion of a predisposition to future disease.

Abstract: Virus-transformed and tumor ceils often show a higher chemotactic activity in comparison to normal cells. Here we show that the blind-well chamber assay is an easy and rapid procedure to select cell populations with enhanced chemotactic activity. Collagen-type-specific antibodies have been applied to analyze the efficiency of cell sorting by the chemotaxis assay.