Single cell sorting and cloning.

Cell sorters now allow the selection of cells and other bodies according to a range of quite diverse criteria. The additional refinement that allows the sorting of individual cells based on these criteria has seen application in many fields of research. Single cells may be sorted for microscopy, for culture and for genetic analysis by way of single cell PCR (polymerase chain reaction). In practical terms, in the setting up of an instrument for single cell sorting, there are additional requirements to ensure that each detected event is indeed a single cell or body, that this cell can be reliably sorted via saline droplet, separate from its fellow travelers, that the aiming of the droplet deflection is sufficiently precise to find the target vessel and that the cell will be undamaged on arrival. Among the diverse reported applications of the technique, two fields which have benefited greatly are lymphocyte development and haemopoiesis. In the former case, the analysis of gene rearrangements in lymphocytes, both in the pre- and post-antigenic phases of development, has been enabled by the combined technologies of single cell sorting and PCR. It is argued that such experiments could not have been done without that partnership. In a similar way, the single cell sorting technique has been found to be the perfect way to demonstrate precursor/progeny relationships between haemopoietic cells and, further, to demonstrate rigorously the effects of particular cytokines on the haemopoietic system.

Flow cytometry and cell-separation procedures.

A series of small incremental advances characterize the year's technical developments in flow cytometry, and these now extend the use of the technique to even the molecular level. The increasing application of immunomagnetic-bead separation procedures dominates the developments in other cell-separation procedures.

Plasmodium falciparum: rapid quantification of parasitemia in fixed malaria cultures by flow cytometry.

A rapid and sensitive method is described for the determination of parasitemia in Plasmodium falciparum cultures using the fluorescence activated cell sorter and DNA-binding fluorochrome, 33258 Hoechst. Conditions were selected to permit its application to the screening of assays with numerous samples. Parasites suspended in culture medium were mixed with an equal volume of aqueous fixative (10% w/v formaldehyde, 4% w/v D-glucose in Tris-saline pH 7.3), stained in a 20 microM final dye concentration, and analyzed with the cell sorter after dilution in Tris-saline. Centrifugation and washing steps were avoided throughout. Close correspondence was obtained between the estimated and actual parasitemia, and fluorescence intensities of infected erythrocytes permitted distinction between ring and schizont stages of the parasites. The ability to store, transport, or assay material rendered not infectious by fixation, and the relative simplicity of this technique are major improvements to methods described previously using living parasites. Reanalysis of fixed material permits reference standards to be used with each assay.

A fast cell sampler for flow cytometry.

A simple device has been developed for delivering samples into a flow cytometer. Designed with economy, simplicity, and flexibility in mind, this device, having only one moving part, can be used for sample volumes as small as 20 microliter, for virtually any form of cell sample container, and for a wide range of cell concentrations. It consists essentially of a lever-operated disc valve that allows the cell sample to be loaded into a loop of tubing and then to be injected into the cytometer nozzle under pressure from a saline source. The sampler has lifted the maximum analytical throughput of a FACS II cell sorter to better than 120 samples per hour.

Fc receptors on mouse neutrophils and eosinophils: antigenic characteristics, isotype specificity and relative cell membrane density measured by flow cytometry.

The antigenic characteristics, isotype specificity and density of Fc receptors (FcR) on mouse neutrophils and eosinophils were studied with the aid of the rat monoclonal antibody 2.4 G2 to the mouse macrophage FcR (Unkeless, 1979). This MAb was tested for its reactivity with mouse neutrophil and eosinophil FcR, and for its ability to block the binding of sheep erythrocytes (E) coated with mouse antibodies of different isotypes to granulocytes. The use of E conjugated with fluorescein isothiocyanate (FITC) allowed an objective read-out by flow cytometry. The MAb 2.4.G2 reacted with both neutrophil and eosinophil FcR, blocking the binding of E coated with mouse IgG1, IgG2a and IgG2b in a dose-dependent manner. Blocking was specific, since it did not occur with any of several control MAb of the same rat isotype (IgG2b) as 2.4.G2. Furthermore, the binding to E through the granulocyte receptor for complement (C) was unaffected. IgG3 was unable to promote binding of E to either neutrophils or eosinophils, although it induced high levels of binding to macrophages. These results show that: (i) neutrophil, eosinophil and macrophage FcR have antigenic similarities; (ii) neutrophils and eosinophils, in contrast to macrophages, either have a common FcR for IgG1, IgG2a and IgG2b, or have different FcR for these isotypes which share the antigenic determinant recognized by 2.4.G2; (iii) in contrast to macrophages, neutrophils and eosinophils lack the FcR for IgG3. The MAb 2.4.G2 was used in an indirect immunofluorescence assay monitored by flow cytometry to measure the relative FcR density on neutrophils and eosinophils. This assay showed that neutrophils possess about 65% more FcR than eosinophils on a cell-for-cell basis, providing an explanation for the higher binding of neutrophils to IgG-coated particles at suboptimal antibody concentrations.

Phenotypic diversity of cloned lines of Leishmania major promastigotes.

In vitro cultured promastigotes of virulent (V) and avirulent (A) cloned lines of Leishmania major, and the parental isolate LRC-L137, were examined with respect to morphology, cell size, growth rate, and apparent DNA content. Growth rates of all lines were comparable and both virulent (V121, LRC-L137) and avirulent parasites (A12, A52, A59) exhibited a progressive decrease in apparent DNA content with time in culture, as measured by incorporation of Hoechst Dye 33342. The four cloned lines and the parental isolate showed differences in the content of morphological variants and in the mean body length. Morphologically, there were similarities between A12 and A52 and between A59 and V121. Promastigote populations were also examined for the expression of the target antigen of a previously characterized monoclonal antibody, WIC-79.3. This antibody binds to a membrane antigen that is also present in culture supernatants of Leishmania of A1 serotype. Three different assays with culture supernatants all showed that V121, A59, and A12 were high producers with LRC-L137 and A52, low producers. Similar variation in expression of the 79.3 target antigen was detected in intact organisms of the various lines by immunofluorescence with flow cytometry. No simple correlation was found between the expression or release of the WIC-79.3 target antigen and virulence. The virulence or avirulence of all cloned lines for BALB/c mice remained stable. The data are discussed in terms of differentiation stages of L. major promastigotes and the continuing search for morphological and biochemical markers of virulence.

Activation of granulocyte cytotoxic function by purified mouse colony-stimulating factors.

Highly purified mouse colony-stimulating factors (CSF) were tested for their effect on neutrophil cytotoxic function in a homologous antibody-dependent cell-mediated cytotoxicity (ADCC) assay in which TNP-coupled mouse thymoma cells coated with mouse anti-TNP antibodies were used as targets, and purified normal mouse bone marrow neutrophils or induced peritoneal neutrophils were used as effector cells. Biochemically pure granulocyte-macrophage (GM)- and granulocyte (G)-CSF enhanced the cytotoxic activity of neutrophils obtained from both sources, allowing them to kill target cells at low antibody concentrations. Furthermore, GM- and G-CSF showed an additive effect, suggesting either the presence of separate receptors for GM- and G-CSF or of separate subsets of neutrophils. Induced peritoneal neutrophils showed a higher level of basal cytotoxic activity than did bone marrow neutrophils, suggesting neutrophil activation in vivo, but both reached similar levels of cytotoxicity upon maximal stimulation with CSF. In addition, CSF was found to be cross-reactive between mouse and human species in their enhancement of neutrophil cytotoxicity. By testing purified mouse CSF on human neutrophils, it could be shown that G-CSF and GM-CSF are functionally distinct molecules, because only G-CSF enhanced ADCC by human neutrophils. These experiments show that the purified factors that control the production of neutrophils by progenitor cells in vitro also activate differentiated neutrophils to carry out their cytotoxic activity in a more effective manner.

Eosinophil activation by colony-stimulating factor in man: metabolic effects and analysis by flow cytometry.

Substantial increases in the killing capacity of human eosinophils after in vitro incubation with human placental conditioned medium (HPCM), a standard source of colony-stimulating factor (CSF), have recently been described. In this article, the interaction between HPCM and purified human eosinophils is analyzed by flow cytometry and by effects on iodination, superoxide production, and protein synthesis. HPCM increased the intensity of natural eosinophil autofluorescence (aFlu) (460 nm) after the absorption of ultraviolet light (360 nm) in a manner that was both time and dose dependent. Measured in arbitrary units, eosinophil aFlu was 72 +/- 7.3 (arithmetic mean +/- SEM) and 121 +/- 3.2 after 18-hr incubations in the absence or presence of HPCM, respectively. The activity in HPCM responsible for these changes cochromatographed on Ultrogel AcA44 columns with CSF and with the less hydrophobic variant of CSF (CSF-alpha) on phenyl Sepharose. Mouse spleen, but not mouse lung, conditioned medium was also active on human eosinophils in this assay. Both CSF-alpha and mouse spleen conditioned medium also contain eosinophil colony-stimulating activity (CSA), whereas inactive CSFs with no effect on mature eosinophils, CSF-beta, and mouse lung conditioned medium also lack eosinophil CSA. CSF-alpha stimulated superoxide production of resting eosinophils (from 0.03 +/- 0.03 to 0.47 +/- 0.08 nmole cytochrome-c reduced/10(5) eosinophils) and of eosinophils incubated with preopsonized zymosan (from 0.15 +/- 0.06 to 0.73 +/- 0.07). It also stimulated iodination by resting eosinophils (from 0.76 +/- 0.16 to 2.60 +/- 0.72 nmoles l/10(7) eosinophils/hr) and of eosinophils incubated with preopsonized zymosan (from 7.52 +/- 2.08 to 29.8 +/- 1.32). In contrast, CSF-beta was inactive in these assays. CSF-alpha also stimulated, between 2- and 15-fold, the new protein synthesis of eosinophils. Thus, substances that stimulate the differentiation of progenitor cells into eosinophils also interact with peripheral mature eosinophils, and the activation of postmitotic cells may be a physiologic role of CSF-like molecules.

Separation of stages of Plasmodium falciparum-infected cells by means of a fluorescence-activated cell sorter.

Plasmodium falciparum parasites from long-term in vitro culture have been labeled with the DNA-binding dye Hoechst 33258. After labeling, parasitized cells have been successfully analyzed and sorted, using a fluorescence-activated cell sorter, into populations of uninfected, singly infected, and multiply infected cells.

Isolation of mouse bone marrow neutrophils by light scatter and autofluorescence.

Murine bone marrow and blood cells have been analyzed and fractionated using an automated FACS II cell sorter. Using visible light scattered in the direction of (0 degrees) and perpendicular to (90 degrees) the laser beam it was possible to enrich for neutrophils (84%), immature myeloid cells (47%), and monocytes (78%). The enrichment for neutrophils was improved to 92% by using the light scattered by ultraviolet laser light (ca.360 nm). The autofluorescence at these wavelengths proved useful for obtaining further enrichment (to 97%). Indeed, three parameter sorting with 0 degrees and 90 degrees light scatter as well as autofluorescence also allowed the separation of lymphocytes (95%) and immature myeloid cells (89%). The same procedures could be applied for the isolation of neutrophils from mouse peripheral blood.

Differential expression of lectin receptors during hemopoietic differentiation: enrichment for granulocyte-macrophage progenitor cells.

Molecular changes occur at the surface of hemopoietic cells during differentiation from progenitor cells to mature granulocytes and macrophages. The differential expression of surface carbohydrate residues has been probed using lectins and the results used to purify normal mouse granulocyte-macrophage progenitor cells. Ten different lectins were screened for selective interaction with mouse hemopoietic colony-forming cells (CFCs), using agglutination or a quantitative analysis of the number of fluoresceinated lectin molecules bound per cell using a fluorescence activated cell sorter (FACS). Pokeweed mitogen (PWM), Helix pomatia agglutinin (HPA), soybean agglutinin (SBA), and peanut agglutinin (PNA) preferentially bound to CFCs so that it was possible to enrich 4 to 10-fold for these progenitor cells by sorting for the highly fluorescent cells. Further analysis of the low and high angle light scattering characteristics of the CFCs indicated that these cells were polydisperse, but could be enriched ten-fold by selecting for cells with high intensity low angle (0 degrees) scatter and low intensity high angle (90 degrees) scatter. PWM gave the best enrichment (10 to 15-fold) for adult bone marrow CFCs, for CFCs from fetal sources (fetal liver, fetal blood), and for CFCs from the spleens of mice injected previously with outer membrane lipoprotein from E. coli. Three parameter sorting for CFC using the FACS (low angle scatter, high angle scatter, and PWM-fluorescence) resulted in large enrichment factors (16 to 50-fold) for CFCs from all the above sources. Over 7% of the cells sorted from bone marrow, 10% of the cells sorted from post-lipoprotein spleen, and 28% of the cells sorted from fetal peripheral blood were hemopoietic CFCs. Ninety percent of the cells in these fractions had the morphology of blast cells or myelocytes. Thus, it was possible to identify the morphological characteristics of the hemopoietic progenitor cells. Screening of other developmental systems using quantitation of fluorescence with lectins should prove of general value for the purification of selected differentiation states.

Cytotoxic and fluorescent assays for thymocyte subpopulations differing in surface thy-1 level.

A number of analytical techniques for distinguishing and separating the "high theta" "low theta" subpopulations of mouse thymocytes have been compared. A differential cytotoxic assay was compared to a quantitative immunofluorescent assay on individual cells using flow cytofluorometry and cell sorting. Conventional anti-Thy-1 antisera were compared with a monoclonal IgM anti-Thy-1. The monoclonal reagent greatly improved both types of assay, eliminated a number of artifacts and allowed either procedure to be used to give a clear distinction, based on Thy-1 level, between the two subpopulations. The distribution of Thy-1 on thymocytes is bimodal, rather than continuous. These separate "high theta" and "low theta" categories each includes a population a population of dividing cells.

Plasmodium berghei - infected red cells sorted according to DNA content.

A cell-sorting method is described for the analysis and separation of red blood cells in Plasmodium berghei-infected mouse blood based on their DNA content. This method involves a selective uptake of the bis-benzimidazole dye 33258 Hoechst, a DNA-binding dye, by red blood cells containing parasites. Infected blood is incubated at 37 degrees C with the dye then washed at 4 degrees C to remove unbound dye. Uninfected cells are then non-fluorescent at the characteristic wavelengths for 33258 Hoechst excitation and emission, whereas parasitized cells display fluorescence intensities in approximately direct proportion tothe number of parasite nuclei (i.e. amount of parasite DNA) within the cell and can be sorted accordingly. Providing cells were incubated in a complex nutrient medium during dye uptake at 37 degrees C, the sorted parasite-infected cells produced lethal P. berghei infections when injected into BALB/c mice. The dye-labelling technique is simple and sufficient red blood cells at various stages of infection can be collected for biochemical or immunochemical studies by cell sorting.

Mechanisms of clonal abortion tolerogenesis. II. Clonal behaviour of immature B cells following exposure to anti-mu chain antibody.

This paper uses B-lymphocyte cloning methods to quantify the effects of anti-mu chain antibody on immature and mature B cells. Nude mouse spleen lymphocytes were incubated with various concentrations of sheep anti-mouse mu chain antibody for times varying from 10 min to 24 h. They were then washed and plated in the agar B-cell colony formation assay. Five to six days later, control B cells had developed into colonies with a plating efficiency of about 5%. B cells from newborn mice pretreated with anti-mu yielded fewer colonies. Remarkably low concentrations sufficed to inhibit subsequent mitogenesis. For example, 3 microgram/ml acting for 1 h or 0.1 microgram/ml acting for 24 h gave greater than 50% inhibition. Adult B cells were about thirty-fold more resistant to negative signalling. Immature cells become more profoundly inhibited as anti-mu treatment was prolonged. Anti-Ia or anti-H2 antibodies, in the absence of complement, did not deliver a negative signal. Anti-mu pretreatment also reduced the capacity of immature B cells to form clones of anti-hapten antibody-forming cells in a liquid microculture system where the triggering stimulus was a T-cell independent antigen. Mature 'T-independent' B cells were not inhibited. Populations of hapten-specific B cells prepared by the hapten-gelatin method were investigated in the agar cloning system. Pretreatment of immature cells with anti-mu reduced their capacity to form colonies, this subpopulation of cells behaving like unfractionated B cells. Furthermore, hapten-HGG delivered a negative signal also. Mature hapten-specific cells or unfractionated immature spleen cells formed normal numbers of colonies following hapten-HGG treatment. Overall, the studies support the view that anti-mu antibody and hapten-HGG deliver strong negative signals to immature but not mature cells with appropriate receptors. The value of anti-mu as a model, universal tolerogen was supported. Fluorescence-activated cell sorter (FACS) analysis was performed to study the relationships between functional inhibition and Ig receptor modulation. We confirmed that the IgM receptors of immature B cells are more readily modulated by anti-mu antibody than those of mature cells. Furthermore, the receptor regeneration could be partially inhibited amongst immature but not mature B cells. There was not a close quantitative relationship between the degree of modulation and the degree of functional inhibition. The results did not support the view that irreversible receptor modulation as such was the cause of functional inhibition.

Plasmodium-infected blood cells analyzed and sorted by flow fluorimetry with the deoxyribonucleic acid binding dye 33258 Hoechst.

Red cells from Plasmodium berghei infected mouse blood can be sorted on the basis of their DNA content with the bisbenzimidazole dye 33258 Hoechst. The optimal conditions for dye uptake have been established and with these conditions uninfected cells are nonfluorescent and can be completely separated from infected cells which exhibit fluorescence in almost direct proportion to the number of parasite nuclei (i.e. DNA) they contain. The number of fluorescent cells detected and their fluorescence intensity is shown to be dependent on the dye concentration and the incubation medium being used. At least a proportion of the infected cells sorted from each fluorescence peak in the cell distribution retain their infectivity in vivo with some, but not all, conditions of labeling. This technique is being used to separate minor cell populations from infected blood for biochemical and immunochemical analyses and to screen human samples for malaria infected cells.