Abstract: The relationship between surface antigen expression and function in the long-term allospecific T cell line C.C3.11.75 was examined by flow microfluorometry, antiserum plus complement depletion and cell sorting. T cells of the line expressed Lyt-1, but little or no Lyt-2 antigens. Proliferation to cells bearing Iak determinants, generation of T cell-replacing nonspecific helper factor in response to Iak-positive cells and the killing of Iak-positive targets were dependent only on Ly-1 cells. No obvious heterogeneity was found in this cell line despite its disparate functional activities. The fact that Lyt-2 molecules need not be present for killing directed against Ia determinants indicates that such molecules are not obligatory for the induction or delivery of killing and raises the question of what the role of Lyt molecules in T cell recognition or function might be.

Abstract: A convenient, preparative scale, nonlytic separation of mouse T lymphocytes into Lyt2.2+ and Lyt2.2- populations is reported. Immunoglobulin-negative (Ig-) spleen cells, Ig- lymph node cells, and peanut lectin-unagglutinated (PNA-) thymocytes were incubated under sterile conditions at 0 degree C with monoclonal mouse antibody to the Lyt2.2 T cell differentiation antigen. The antibody-treated cells were washed and placed in polystyrene tissue culture dishes that had been precoated with antibody to mouse Ig. Nonadherent populations were depleted to Lyt2.2+ cells and were essentially devoid of cytotoxic T lymphocyte precursors (CTLp), but contained helper activity for in vivo T-dependent IgM, IgG and IgA antibody formation. Adherent cell populations were enriched for Lyt2.2+ cells and for CTLp. The graft-vs.-host activity of the separated, adherent (Lyt2.2+) and nonadherent (Lyt2.2-) cells in the Simonsen spleen assay in neonatal (C57BL/6 x BALB/c)F1 mice was less than of unfractionated cells, but the activity of remixed Lyt2.2+ plus Lyt2.2- cells was higher than the sum of the contributions of these cells tested separately, and equal to that of the unfractionated cells. PNA- thymocytes were also separated into Ly2.2+ and Lyt2.2- populations by fluorescence-activated cell sorting. Nonlytic separation allows the recovery of the Lyt1+2+ population, which is lost in cytotoxic elimination experiments. Under the conditions described for the plate separation, the purity of the separated cells and recovery of activity approaches that of cells separated by sorting. Therefore, the plate separation offers a convenient alternative to fluorescence-activated cell sorting when large numbers (i.e. up to 5 x 10(7) positively selected cells) are needed, as in studies of in vivo cell-mediated immune reactions.

Abstract: An immunofluorescence method for monitoring DNA synthesis in single cells has been developed for flow cytometry. With antiserum which is specific for 5-bromodeoxyuridine (BrdUrd) and a second fluorescent label, BrdUrd-incorporation pulses of 30 min are detectable. The fluorescence intensity of the incorporated BrdUrd, as determined by immunofluorescence, is related to the amount of BrdUrd incorporated, as shown by isotopic methods and cell sorting. Thus, the technique may be applicable to determining rates of replication per cell. Multiple samples of as few as 1 × 105 cells can be fixed, hydrolyzed and treated with the anti-BrdUrd antiserum. Nuclear-bound IgG is localized by fluorescein-labeled avidin-D. Since the technique uses whole cells, other parameters such as light scatter and DNA content can be simultaneously monitored so that cohorts of “labeled” cells can be followed through the cell cycle.

Abstract: We obtained Thy-1-positive cells directly from growing methylcholanthrene-induced (MCA-1510) sarcomas using fluorescence-activated cell sorting, then cultured these lymphocytes in medium containing Interleukin-2 and tested their activity in vivo against various MCA-sarcoma lines with the Winn assay. We found that cultured T cells from small MCA-1510 tumors (17 days after transplantation) significantly inhibited the growth of that particular sarcoma, but not of three other MCA-tumor lines tested, while cultured T cells from large MCA-1510 sarcomas (41 days after transplantation) significantly enhanced the growth of that tumor, but not of an unrelated tumor, MCA-1460. The former cells were primarily Lyt-1+, 2+ while the latter were primarily Lyt-1−, 2+.

Abstract: A bacterial neutral protease was used to disperse KHT solid tumors into single cell suspensions suitable for routine cell kinetic analysis by flow cytometry and for clonogenic cell survival. Neutral protease disaggregation under conditions which would be suitable for routine tumor dispersal was compared with a trypsin/DNase procedure. Cell yield, clonogenic cell survival, DNA distributions of untreated and drug-perturbed tumors, rates of radioactive precursor incorporation during the cell cycle, and preferential cell cycle phase-specific cell loss were investigated. Tumors dispersed with neutral protease yielded approximately four times more cells than those dispersed with trypsin/DNase and approximately a 1.5-fold higher plating efficiency in a semisolid agar system. Quantitative analysis of DNA distributions obtained from untreated and cytosine-arabinoside-perturbed tumors produced similar results with both dispersal procedures. The rates of incorporation of tritiated thymidine during the cell cycle were also similar with neutral protease and trypsin/DNase dispersal. Preferential phase-specific cell loss was not obseved with either technique. We find that neutral protease provides good single cell suspensions of the KHT tumor for cell survival measurements and for cell kinetic analysis of drug-induced perturbations by flow cytometry. In addition, the high cell yields facilitate electronic cell sorting where large numbers of cells are oftenmore » required.« less

Abstract: We used a bacterial neutral protease to disperse KHT solid tumors into single cell suspensions suitable for routine cell kinetic analysis by flow cytometry and for clonogenic cell survival. Neutral protease disaggregation under conditions which would be suitable for routine tumor dispersal was compared with a trypsin/DNase procedure. Cell yield, clonogenic cell survival, DNA distributions of untreated and drug-perturbed tumors, rates of radioactive precursor incorporation during the cell cycle, and preferential cell cycle phase-specific cell loss were investigated. Tumors dispersed with neutral protease yielded approximately four times more cells than those dispersed with trypsin/DNase and approximately a 1.5-fold higher plating efficiency in a semisolid agar system. Quantitative analysis of DNA distributions obtained from untreated and cytosine-arabinoside-perturbed tumors produced similar results with both dispersal procedures. The rates of incorporation of tritiated thymidine during the cell cycle were also similar with neutral protease and trypsin/DNase dispersal. Preferential phase-specific cell loss was not observed with either technique. We find that neutral protease provides good single cell suspensions of the KHT tumor for cell survival measurements and for cell kinetic analysis of drug-induced perturbations by flow cytometry. In addition, the high cell yields facilitate electronic cell sorting where large numbers of cells are often required.

Abstract: Monoclonal anti-Thy-1.1 and anti-Thy-1.2 antisera selected for complement-dependent cytotoxicity have high cytotoxic and binding titers on thymocytes and peripheral T cells of mouse strains bearing the appropriate Thy-1 allele. The effect of both anti-Thy-1.1 and anti-Thy-1.2 monoclonal antisera plus complement on cytotoxic T cell effectors is to abrogate their activity. On the functional activity of precursor cytotoxic T cells, monoclonal antisera against the two alleles have different effects: anti-Thy-1.2 plus complement removes precursor activity of Thy-1.2-bearing strains, including (Thy-1.1 × Thy-1.2)F1 heterozygotes. In contrast, six different anti-Thy-1.1 monoclonals, including four of the IgM class and two of the IgG class, failed to remove cytotoxic precursor activity from the splenic T cells of AKR, A. Thy-1.1 or (CBA × AKR)F1 mice. Analysis by fluorescence-activated cell sorting of in vitro cultured AKR spleen cells shows that Thy-1.1 antigen appears on the cell surface during the five-day culture period.

Abstract: In order to test the hypothesis that the progressive layering and differentiation of cell types during the development of the neural retina are associated with cell surface alterations we have separated distinct cell populations from the 14-day embryonic chick retina. Cells of these populations have been shown to differ in associative behavior and intramembrane particle content. We now report that these cells differ in cell surface glycoproteins. Proteins were labeled with two different extrinsic labels and one metabolic label. We used enzymatic transfer of galactose from UDP-gal to cellular acceptors, and borotritide reduction after galactose oxidation as extrinsic labels. Glucosamine incorporation was used as the metabolic label. In all these cases, we were able to identify bands on electrophoretic gels which were unique to individual populations.

Abstract: Lavaged lung cells from normal rabbits and rabbits given hydrochloric acid intratracheally were analyzed using flow cytometry techniques. The rabbit alveolar macrophage was characterized as a cell with relatively high light scatter and high intrinsic autofluorescence compared with the lung neutrophil with its lower light scatter and autofluorescence. The flow cytometry characteristics of these 2 cell populations were confirmed with light microscopy after cell sorting. A flow cytometry analysis of alveolar macrophage phagocytosis using fluorescent latex particles was applied to normal rabbit alveolar macrophages. These studies showed that flow cytometry can be used to study the morphologic features and function of rabbit lavage lung cell populations and may be ultimately useful in the analysis of human lung cells.

Abstract: The immunomodulating 2-cyanaziridine derivatives BM 12.531 (azimexone) and BM 41.332 have no effects on the total amount of T-lymphocytes in the spleen of mice but ingrease dose-dependently the percentage of Ly1−-, 2+, 3+ T-lymphocytes (killer/suppressor) and decrease the percentage of Ly1+, 2−-, (helper) cells. These investigations were carried out by means of specific monoclonal antibodies and fluoreqcence-activated cell sorting. The increase in Ly2+-cells is mainly due to increased suppressor activity.

Abstract: The properties of human lymphocyte fractions isolated either by sheep red cell(E) rosetting or by fluorescence-activated cell sorting after staining with UCHT1 monoclonal anti-T cell antibody have been compared. Two populations of E+ cells with very different phenotype and function have been identified. E+/UCHT1+ cells respond well to the T cell mitogens phytohemagglutinin and concanavalin A and provide help for an in vitro specific antibody response. They can also suppress the antibody response of allegeneic peripheral blood mononuclear cells. In contrast, the E+/UCHT1- population, which has no other markers characteristic of T cells, fails to respond to mitogens or to provide help or suppression for an antibody response. These cells, however, are highly active natural killers. They possess Fc gamma receptors and have a characteristic staining pattern of nonspecific esterase enzyme activity. It is concluded that not all cells capable of forming E rosettes are thymus-processed cells and that this heterogeneity can be revealed by staining with the monoclonal anti-T cell reagent UCHT1.

Abstract: Previous studies using semi-solid agar cultures of acute myeloid leukaemia (AML) cells have shown that proliferative capacity (clone size potential) and the degree of sensitivity of clone-forming cells to the specific granulocyte-macrophage regulatory colony-stimulating activity (CSA) are closely linked AML cell properties. The purpose of this study was to determine whether this association is confined to AML cells or whether the linking of these two properties in AML represents retention of an association occurring in normal granulocyte-monocyte progenitor cell populations. Bone marrow cells from normal donors were studied using four independent techniques to enrich for clonogenic cells with different clone size potentials (equilibrium density centrifugation, adherence to microcarrier beads, osmotic lysis and fluorescence-activated cell sorting). It was shown that mean clone size was directly related to the mean CSA threshold (amount of CSA required to stimulate 50% of the cells to clone formation). Further studies (including analysis of the kinetics of production of clonogenic cells with different clone size potentials in suspension culture) suggested that these two properties were linked to differentiation, with proliferative capacity decreasing and sensitivity to CSA increasing as cells differentiate down the granulocyte-monocyte pathway.

Abstract: The unexpected discovery that la-like (HLA-DR) antigens1–3 in humans were present on blast cells from acute myeloblastic leukaemia4,5 led to the finding that normal granulocytic progenitors, in contrast to their mature descendents, also expressed HLA-DR antigens. Thus, anti-Ia sera stain a proportion of myeloblasts in normal bone marrow6,7, inhibit myeloid progenitor (CFU-GM) colony formation in the presence of complement8 and can be used to label and separate CFU-GM on a fluorescence-activated cell sorter (FACS)9. Winchester et al. subsequently reported that erythroid progenitors (BFU-E and CFU-E) were also inhibited or killed by anti-Ia (p28,37) and complement10. These observations raised the possibility that HLA-DR (or presumptive I-region equivalent) products might have a regulatory role in early haematopoiesis. We have now analysed HLA-DR and HLA-ABC antigen expression on normal erythroid progenitors using monoclonal antibodies to non-polymorphic determinants and fluorescence-activated cell sorting. In parallel experiments, we tested a monoclonal antibody to glycophorin, a well defined erythroid-speciflc cell-surface membrane glycoprotein11. We report that HLA-DR, HLA-ABC and glycophorin are all expressed at various stages during erythroid differentiation.

Abstract: A marked gradient of Adriamycin uptake in cells of tissue-like multicell spheroids in vitro has been demonstrated by fluorescence photomicroscopy and flow microfluorimetry techniques. As expected, this gradient led to an increased net survival for cells from Adriamycin-treated spheroids relative to monolayers and markedly greater clonogenicity of central spheroid cells than external cells selected by fluorescence-activated cell sorting. Growth of cells as spheroids seemed to impart an additional degree of drug resistance relative to cells grown as monolayers, in that equal toxicity required greater intracellular fluorescence (and thus more Adriamycin) for the spheroid cells. The flow cytometry techniques thus provide a mechanism for quantification of Adriamycin penetration into spheroids and provide a method for selection of cells from various depths within the spheroid.