Biomedical subjects
Publications and source records attributed to F L Battye.
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.
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 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.
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.
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.
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.
A two-step sorting procedure with the fluorescence-activated cell sorter (FACS) is described to select T cell hybridomas from cell populations obtained from the fusion of activated T cells and T- lymphoma cells. Cells are first sorted on the basis of differences in cell-surface antigens. After a period of growth in culture, the cells are then stained with the DNA-labeling dye dis-benzimidazole 33258 Hoechst and sorted on the basis of their DNA content. From 15 to 25% of the resulting cell clones, growing out of limiting dilutions, were hybrid lines as judged by chromosome analysis.
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.
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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.
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.
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Explore the source record for details and available documents.
Explore the source record for details and available documents.
Mouse bone marrow cells were fractionated on columns of wheat germ agglutinin-Sepharose 6MB (WGA-Sepharose) and conditions established for specific binding and cell enrichment in the eluted fractions. A small proportion (7%) of the cells applied did not bind to the column and 60% of these were lymphocyte-like cells. Twice as many cells were eluted from the WGA-Sepharose column using N-acetyl-D-glucosamine (GlcNAc), and 80% of these cells were polymorphonuclear granulocytes and metamyelocytes. These cells were only released from the matrix in the presence of GlcNAc at high buffer flow rates (greater than 4 ml/min). Approximately 3 x 10(8) bone marrow cells bound to 1 ml of WGA-Sepharose. At least 5 min were required for 90% binding of the cells and elution of the cells with GlcNAc required nearly 20 min at 24 degrees. At 37 degrees the elution of cells with ClcNAc was much faster (less than 5 min) and a much larger percentage of cells (40-50%) was specifically eluted. Sodium azide (0.02%, w/v) did not prevent cells binding to WGA-Separose or alter the distribution of cells eluted by GlcNAc, but did slightly increase cell yields. Elution of cells with different concentrations of GlcNAc gave fractions enriched in different cell types. Analysis of cell fraction with a fluorescence-activated cell sorter showed that the lymphocyte subpopulation which failed to bind to WGA-Sepharose had been depleted of cells with a high density of immunoglobulin on their surface. The lymphocytes with a high density of surface immunoglobulin were recovered in the cells released from WGA-Sepharose using mechanical agitation.