Showing papers on "Cell sorting published in 1989"
TL;DR: Transfected cells with cDNAs coding for two calcium-dependent CAMs of different specificity, the liver CAM (L-CAM) and the structurally related molecule N-cadherin, could distinguish at least eight cell lines by their behavior in sorting-out assays, suggesting that qualitative and quantitative differences in the expression in vivo of a relatively small number of CAMs can lead to a large variety of patterns among cell collectives and their borders during tissue formation.
Abstract: Cell adhesion molecules (CAMs) are cell surface glycoproteins that may play a variety of roles in morphogenesis and histogenesis, particularly in defining borders of discrete cell populations. To examine the influence of CAM expression on such cell segregation events in vitro, we have transfected cells with cDNAs coding for two calcium-dependent CAMs of different specificity, the liver CAM (L-CAM) and the structurally related molecule N-cadherin. The cDNAs were introduced separately or together into murine sarcoma S180 cells, which normally do not express these molecules, to produce cell lines denoted S180L, S180cadN, and S180L/cadN, respectively. A number of cell lines of each type were produced that differed in their levels of CAM expression. In adhesion assays, S180L and S180cadN cells aggregated specifically via their respective CAMs, and S180L cells did not appear to adhere to S180cadN cells. Cells expressing high levels of each CAM aggregated more rapidly than cells expressing low levels. Segregation between two cell types occurred when they expressed CAMs of different specificity or different levels of the same CAM. S180L and S180cadN cells both sorted out from untransfected cells, and cells expressing high levels of either L-CAM or N-cadherin segregated from cells expressing low levels of the same CAM; in all cases segregation was inhibited by antibodies specific for the transfected CAM. S180L cells sorted out from S180cadN cells, but this segregation was inhibited only when antibodies to both CAMs were applied together. Doubly transfected S180L/cadN cells also sorted out from S180L cells and from S180cadN cells, and the process was inhibited by antibodies to the unshared CAM (N-cadherin or L-CAM, respectively). Cytochalasin D and nocodazole inhibited sorting-out, consistent with the probable role of microfilaments and microtubules in cell movement and in accord with evidence that the action of these CAMs depends on interactions with cortical cytoplasmic components. Using cDNAs for only two CAMs in these studies, we could distinguish at least eight cell lines by their behavior in sorting-out assays. This suggests that qualitative and quantitative differences in the expression in vivo of a relatively small number of CAMs can lead to a large variety of patterns among cell collectives and their borders during tissue formation.
247 citations
TL;DR: In this article, surface expression of GPIIb-IIIa, measured by fluorescent activated cell sorting or by surface labeling, required cotransfection of both subunits, while surface expression was not detected when the subunits were transfected individually.
180 citations
Journal Article•
TL;DR: The effects of CT on normal B cells and a clonal B cell line indicate that CT induces substantial numbers of mIgM+ cells to undergo isotype differentiation intomIgG+ or mIGA+ B cells.
Abstract: Cholera toxin (CT) is a powerful oral immunogen and adjuvant that elicits strong IgG and IgA antibody responses In our study we investigated whether this property of CT was associated with an effect on B cell isotype differentiation Initially, we determined the effect of CT on normal LPS-induced Peyer's patch B cells and found that whereas CT is strongly inhibitory of IgM production, it increases by approximately three-fold the number and frequency of IgG- and IgA-producing cells Subsequently, using cell sorting technology, we demonstrated that CT acts on membrane (m)IgM+, mIgG/mIgA- B cells rather than mIgG/mIgA+ B cells In addition, we showed that CT does not cause selective inhibition of mIgM, or enhancement of mIgG/mIgA B cell proliferation In parallel studies we determined the effect of CT on the differentiation of a clonal B cell population, CH12LX cells, ie, a population comprised mainly of mIgM+ cells (98%) admixed with a small subpopulation of mIgA+ cells (2%) Here we found that CT (in the absence of LPS) causes a rapid decrease (24 h) in the intensity of mIgM expression as well as a marked increase in the size of the subpopulation expressing mIgA In addition, we found that CT (in the presence of LPS), inhibits CH12LX IgM production while increasing the absolute number and frequency of IgA-producing cells In contrast, CT inhibits IgA production by CH12LXA2 cells, a subclone of CH12LX cells that bears only IgA Finally, we demonstrated that CT is equally inhibitory of the proliferation of CH12LX cells and CH12LXA2 cells Taken together, these effects of CT on normal B cells and a clonal B cell line indicate that CT induces substantial numbers of mIgM+ cells to undergo isotype differentiation into mIgG+ or mIgA+ B cells In a final series of studies we showed that the effect of CT on isotype differentiation was mimicked by the B subunit of CT, ie, the subunit that does not activate intracellular adenylate cyclase; thus the induction of isotype differentiation by CT is not mediated by a perturbation in cAMP level
96 citations
TL;DR: Results show that transfected human DNA sequences determine expression of the (alpha-1,2)fucosyltransferases in the mouse transfectants, that these sequences represent a single locus, and that they are within or linked to specific human restriction fragments identifiable in each secondary transfectant.
92 citations
TL;DR: The development of the flow cytometry and cell sorting technologies provides the ways in which the acquisition of unique information about populations of cells and subcellular organelles, coupled to the manipulation of these populations in a manner not possible using other techniques, can significantly augment the understanding of the molecular organization of living cells.
Abstract: Publisher Summary This chapter discusses the process of development of flow cytometry and cell sorting procedures for the analysis of higher plant cells. The development of the flow cytometry and cell sorting technologies provides the ways in which the acquisition of unique information about populations of cells and subcellular organelles, coupled to the manipulation of these populations in a manner not possible using other techniques, can significantly augment the understanding of the molecular organization of living cells. A fundamental aspect of instrumentation used in flow cytometry and cell sorting centers is based on the optical properties of populations of individual cells suspended within a high-velocity fluid stream. The principle type of measurement made with flow cytometers involves the detection and quantitation of fluorescence. The characteristics and attributes that are unique to plant systems are: (1) the direct isolation of rare mutant or variant types, including those possessing enhanced capabilities for embryogenesis and those exhibiting altered photosynthetic activities, (2) the isolation of mutant or variant gametophytes, such as pollen at the microspore level, and (3) the isolation of transformed protoplasts in vivo .
76 citations
Journal Article•
TL;DR: The concept that neuropeptides, such as enkephalins, have a role in the modulation of the immune response and may participate in the bidirectional communication between the nervous and immune systems is supported.
Abstract: The expression of proenkephalin A (PEA), a neuropeptide-encoding gene, was examined in normal rat lymphocytes. With the use of Northern blot hybridization analysis of total RNA, PEA mRNA was found in normal cells derived from spleen, lymph nodes, and bone marrow. Cell sorting of the two main fractions of B and T cells derived from the spleen revealed that PEA is expressed in normal B cells (sIg+). The expression of PEA mRNA was markedly enhanced after a short incubation (3 h) of cells with LPS or Salmonella typhimurium. This was not the case when these cells were incubated with Con A during the same period of time; whereas, in thymocytes the presence of PEA mRNA was exclusively dependent upon mitogenic stimulus (Con A) and could be detected after 24 h of in vitro incubation. Extracts of cells were also found to contain immune reactive enkephalins, indicating that the PEA mRNA is translated. These results support the concept that neuropeptides, such as enkephalins, have a role in the modulation of the immune response and may participate in the bidirectional communication between the nervous and immune systems.
76 citations
TL;DR: It is demonstrated that the level of target cell class I HLA expression is not sufficient to determine susceptibility or resistance to NK-mediated cytolysis of the classical NK targets K562 and MOLT-4.
Abstract: To test the hypothesis that susceptibility to NK cell-mediated cytolysis varies inversely with the levels of target cell class I HLA expression, NK-susceptible K562 and MOLT-4 target cells have been transfected via electroporation with cloned human class I HLA-A2 and HLA-B7 genes. Stably transfected cells expressing varying levels of cell-surface class I HLA have been selected by fluorescent activated cell sorting and tested for susceptibility to NK-mediated cytolysis by freshly isolated peripheral blood NK cells from nine normal volunteers as well as by cloned human NK effectors and tumor cells from a patient with an NK cell lymphoproliferative disorder. Expression of class I HLA did not alter the susceptibility of K562 or MOLT-4 target cells to NK-mediated cytolysis by any of the effectors tested. In addition, the class I HLA-expressing transfectant cells were identical to mock transfected cells in their ability to compete for lysis in cold target inhibition assays. Treatment of both mock-transfected and class I HLA-transfected K562 cells with IFN-gamma resulted in decreased susceptibility to NK-mediated cytolysis which was independent of the total level of class I HLA expression. These results demonstrate that the level of target cell class I HLA expression is not sufficient to determine susceptibility or resistance to NK-mediated cytolysis of the classical NK targets K562 and MOLT-4.
72 citations
Journal Article•
TL;DR: Histological enrichment, cell sorting, polymerase chain reaction, and direct sequencing of the c-Ki-ras-2 gene revealed mutations in codon 12 were present in both aneuploid and diploid subpopulations of sorted carcinomas, suggesting that these mutations precede ploidy alterations in the progression of these neoplasms.
Abstract: We have analyzed colon carcinomas by a combination of histological enrichment, cell sorting, polymerase chain reaction, and direct sequencing of the c-Ki- ras -2 gene. DNA was chemically extracted from 50-µm sections of paraffin-embedded colon carcinomas and amplified in vitro , and mutations were documented directly by DNA sequencing. Enrichment for tumor cells was obtained histologically and by sorting nuclei on the basis of DNA content differences. Mutations in codon 12 were present in both aneuploid and diploid subpopulations of sorted carcinomas, suggesting that these mutations precede ploidy alterations in the progression of these neoplasms. We have demonstrated the feasibility of utilizing DNA from tissues treated with different fixatives, including methyl carnoys, formalin, and Hollande's solution. This procedure allows one to retrospectively reconstruct the temporal relationship between the occurrence of mutations and sequential morphological changes during tumorigenic progression.
54 citations
TL;DR: It is hypothesized that phagocyte activation and production of cytotoxic reactive oxygen intermediates may contribute to hematotoxicity induced by benzene.
Abstract: Techniques in flow cytometry/cell sorting were used to characterize the effects of benzene and its metabolites on subpopulations of bone marrow cells. Treatment of male Balb/c mice with benzene (88...
44 citations
TL;DR: A retroviral vector encoding the receptor for human colony-stimulating factor-1 (CSF-1) was introduced into murine myeloid FDC-P1 cells which require interleukin-3 (IL-3) for their proliferation and survival in culture and the induction of both the CSf-1-dependent and factor-independent phenotypes depended on expression of the transduced human CSF- 1R.
Abstract: A retroviral vector encoding the receptor for human colony-stimulating factor-1 (CSF-1) was introduced into murine myeloid FDC-P1 cells which require interleukin-3 (IL-3) for their proliferation and survival in culture. Cells expressing the CSF-1 receptor (CSF-1R), selected by fluorescence-activated cell sorting in the continued presence of murine IL-3, formed colonies in semisolid medium and were able to proliferate continuously in liquid cultures containing human recombinant CSF-1. Thus, although they do not synthesize endogenous murine CSF-1R, FDC-P1 cells express the downstream components of the CSF-1 mitogenic pathway necessary for its signal-response coupling. After receptor transduction, slowly proliferating factor-independent variants that produced neither CSF-1 nor growth factors able to support the proliferation of parental FDC-P1 cells also arose. When the human CSF-1R was expressed in FDC-P1 cells under the control of an inducible metallothionein promoter, the frequencies of both CSF-1-responsive and factor-independent variants increased after heavy-metal treatment. In addition, a monoclonal antibody to human CSF-1R arrested colony formation by both the CSF-1-dependent and factor-independent cells but did not affect their growth in response to IL-3. Therefore, the induction of both the CSF-1-dependent and factor-independent phenotypes depended on expression of the transduced human CSF-1R.
33 citations
Journal Article•
TL;DR: In bone marrow-derived long term lymphoid cultures initiated in presence of IL-4, the majority of cells exhibited a more immature phenotype than is usually seen in lymphoid cells grown in Whitlock-Witte type cultures.
Abstract: IL-4 influences the cellular composition of stromal cell dependent long term cultures. In bone marrow-derived long term lymphoid cultures initiated in presence of IL-4, the majority of cells exhibited a more immature phenotype than is usually seen in lymphoid cells grown in Whitlock-Witte type cultures. This immature cell population, lacking the B220 Ag, was purified by cell sorting. When transferred to mixed stromal layers used in conventional lymphoid long term cultures, these cells differentiated into B lineage cells that could be identified by expression of the B220 Ag and surface IgM. Abelson murine leukemia virus-transformed cell lines resulting from this immature cell population express a DJH rearrangement and contained RNA that hybridized with a VJ558 probe, suggesting transcription of germ-line V genes. A culture modification allowed selective proliferation of a non-transformed cell population with characteristics of very immature B lineage cells. The proliferation of these cells was supported by a homogeneous stromal cell line that was propagated with horse serum in presence of IL-4. The lymphoid cells proliferating under those culture conditions expressed the Ag detected by the BP-1 and 6C3 mAb and were Fc gamma RII and Ia-positive. However, more mature B cell markers were lacking. DNA analysis of these cell lines revealed JH rearrangement without evidence for deletion of any member of the DSP-2 family. These cell lines retained their immature phenotype after transfer to mixed stromal layers of Whitlock-Witte type. The mechanisms providing these unique culture conditions initiated by IL-4 in bone marrow stromal cells are discussed.
TL;DR: In this article, a procedure is described to select mutants of Chinese hamster ovary cells that are conditionally defective for the cell-surface expression of integral membrane glycoproteins, including the hemagglutinin (HA) of influenza virus.
Abstract: A procedure is described to select mutants of Chinese hamster ovary cells that are conditionally defective for the cell-surface expression of integral membrane glycoproteins, including the hemagglutinin (HA) of influenza virus. Using a combination of cell sorting and biochemical screening, seven cell lines were obtained that express more cell-surface HA at 32 degrees C than at 39 degrees C. The production of infectious vesicular stomatitis virus, whose growth requires insertion of an integral membrane protein into the plasma membrane, was also temperature conditional in the majority of these mutant cell lines. Five of the lines synthesized apparently normally core-glycosylated HA at the elevated temperature but the protein was neither displayed on the cell surface nor accumulated intracellularly. In these cell lines, little or no terminally glycosylated HA molecules were observed after synthesis at 39 degrees C. By contrast, the core glycosylation of HA and several other integral membrane proteins was abnormal in the remaining two cell lines at both permissive and restrictive temperatures, due to a lesion in a cellular gene(s) that affects the formation of and/or the addition of mannose-rich oligosaccharide chains to newly synthesized polypeptides. Although HA was transported to the plasma membrane at both 32 and 39 degrees C, it did not accumulate on the cell surface at the higher temperature, apparently because of an increased rate of degradation.
Journal Article•
TL;DR: Negative immunomagnetic selection using an anti-7/4, anti-B220 antibody cocktail and second-antibody-coupled Dynabead microspheres to replace flow cytometry results in the highly reproducible and specific enrichment of HPP-CFC, and simultaneous resolution of HPCFC from CFCCSF-1.
TL;DR: Labeling mice with bromodeoxyuridine at various times prior to cell isolation showed that the least-differentiated basal cells cycle more slowly than those at later stages,Data support the concept of a differentiation-related, hierarchical pattern of organization of the proliferative compartment.
TL;DR: Findings indicate that cells other than NK effectors or mature T cells are capable of generating a LAK cell response, and that these cells are different from AK or antigen-specific CTL precursors.
Abstract: Separation of LAK precursor (LAKp) cells (as defined by LAK effector generation after incubation with IL-2 for 7 d) from cells with NK activity/LGL morphology was achieved on Percoll gradients using a longer, slower centrifugation than that used for optimal NK enrichment. mAb were generated using the various Percoll fractions as the immunizing cells and used for separation and depletion studies. Two mAbs DM-1 (IgM,k) and DM-2 (IgM,k) recognizing 2-15% and 15-30% of PBL, respectively, abrogated a large proportion of LAK generative potential after complement depletion, but had little effect on NK or LAK effector activity. Cell sorting experiments indicated that the majority of LAKp cells are found within the DM-1+ population and that DM-1+ cells are not simply an accessory cell required for LAKp generation. Further, these two mAbs do not recognize cells that are responsible for generating cytotoxicity during MLC or co-culture with the PR-1 EBV lymphoblastoid cell line. Western blot analysis indicated that DM-1 and DM-2 recognize a 38,000 and 44,000 dalton moiety, respectively. The frequency of cells bearing these antigens and the intensity of cell surface staining decreased during the 7-d culture period, suggesting that these antibodies recognize determinants found only at the precursor level. These findings indicate that cells other than NK effectors or mature T cells are capable of generating a LAK cell response. These LAK precursor cells share a common differentiation surface antigen and are different from AK or antigen-specific CTL precursors. The possibility exists that these cells are identical to, or include, the NK precursor cell.
Journal Article•
TL;DR: Cell death after MNNG exposure occurred during the second cell cycle after treatment apparently as a consequence of perturbation of DNA replication and the degradation of nuclear DNA.
Abstract: The mechanisms involved in cell death caused by carcinogens that methylate DNA are poorly understood. In this study, the cytotoxicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was studied in exponentially growing T5-1 human lymphoblastoid cells. MNNG exposure killed cells and inhibited proliferation of the remaining viable cells. Reduction in cell viability, which coincided with the accumulation of cells in the late S phase of the cell cycle, was not apparent until the population had completed one doubling. Fluorescence-activated cell sorting of fluorescein diacetate-stained, MNNG-treated cells into live and dead subpopulations revealed that all cycle phases were well represented in the live fraction, whereas the dead fraction consisted primarily of cells with a sub-G1 DNA content. Thus, cell death after MNNG exposure occurred during the second cell cycle after treatment apparently as a consequence of perturbation of DNA replication and the degradation of nuclear DNA.
TL;DR: It was found that BPV-transformed cells expressing a truncated version of gp340/220 still produced it at significant levels after extended passage, and this was in contrast to previous studies that found cells that lost the ability to express this glycoprotein on passage.
Abstract: Summary
Epstein-Barr virus (EBV) membrane antigen glycoproteins gp340 and gp220 are encoded by a single gene. We have inserted this gene into a bovine papillomavirus (BPV) vector and expressed gp340/220 in mammalian cells under the control of the mouse metallothionein promoter. The proteins produced were of similar M
r, showed similar antigenic specificity and were transported to the same subcellular location as the authentic gp340/220. The inclusion of heavy metal ions in the medium had no effect on the levels of gp340/220, which were approximately the same as those found in standard EBV-transformed lymphoblastoid cell lines, e.g. B95-8. Cells that expressed gp340/220 were selected by several rounds of fluorescence-activated cell sorting, but on passage they rapidly lost the ability to express this glycoprotein. In contrast to this we found that BPV-transformed cells expressing a truncated version of gp340/220 still produced it at significant levels after extended passage.
TL;DR: To improve the ability of flow cytometry to detect multidrug-resistant cells, the extent to which cell volume heterogeneity accounts for the variance of intracellular daunorubicin (DNR) content was studied.
Abstract: To improve the ability of flow cytometry to detect multidrug-resistant cells, we studied the extent to which cell volume heterogeneity accounts for the variance of intracellular daunorubicin (DNR) content. For P388 murine or HL-60 human leukemia cells exposed to DNR (1 micrograms/ml, 60 min), log intracellular DNR content varied in direct proportion to log cell volume measured by flow cytometry, with a correlation coefficient of .9. This relationship was confirmed by cell sorting based on intracellular DNR content with subsequent volume determination of the sorted cells. Normalization of intracellular DNR content for cell volume (thus obtaining intracellular DNR concentration) was accomplished by subtracting log cell volume from log intracellular DNR content for each cell. This resulted in a 34% decrease (range 23-58%) in standard deviation compared to DNR content measurements without volume normalization for all cell types tested. Following exposure to DNR (as above), intracellular DNR content of drug-sensitive P388 or HL-60 cells measured by flow cytometry was 12- and 8-fold greater than that of the multidrug-resistant sublines P388/ADR and HL-60/AR, respectively. However, because of the variance of intracellular DNR content, the predictive value of flow-cytometric determination of intracellular DNR content as a discriminant assay for detecting the frequency of drug-resistant cells in a mixed population was acceptable only when the frequency of resistant cells in the population exceeded 10%. In contrast, volume normalization of intracellular DNR content enhanced the ability of the flow-cytometric assay to discriminate resistant cells by 10-fold for P388 cells and 100-fold for HL-60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Journal Article•
TL;DR: The studies suggest that the suppressive effect of SP1 occurs in the absence of certain accessory cells (e.g., monocytes and T-lymphocytes), that this effect may be mediated directly at the level of the HPC, and that this pentapeptide can be considered a candidate modulatory molecule for HPC proliferation.
Journal Article•
TL;DR: The enzyme activities of thymidine kinase (TK), thymidylate synthase (TS), DNA polymerase (DNA-P), dihydrofolate reductase (FH2-R), methionine synthase, and hexokinase (HK) were determined in the obtained cell fractions.
Abstract: Exponentially growing human lymphoblasts (culture LS-2) were separated by cell sorting (FACS II, Becton Dickinson) according to their deoxyribonucleic acid (DNA) content, designating them at particular phases of the cell cycle. Prior to cell sorting the DNA has been fluorochrome-labeled with the Hoechst stain H 33342. Maximum cell enrichments of 94% for G0 + G1 cells, 96% for S cells and 74% for G2 + M cells could be achieved. The enzyme activities of thymidine kinase (TK), thymidylate synthase (TS), DNA polymerase (DNA-P), dihydrofolate reductase (FH2-R), methionine synthase (MS), and hexokinase (HK) were determined in the obtained cell fractions. Although incorporation of 3H-thymidine (3H-dTR) and the 3H-dTR labeling index were significantly inhibited by the dye, no evidence of cell staining's having a significant effect on the enzyme activities was found. The enzyme activities for approximately 100% pure G0 + G1, S, and G2 + M cells were computed. With exception of TK, all the enzymes under study were shown to exhibit activities--although of differing degree--in the G0 + G1, S, and G2 + M cells. No TK activity was shown in G0 and G1 cells; its activity, however, was approximately the same in S and G2 + M cells. This applies likewise for TS which, in contrast to TK, exhibits minor activity in G0 + G1 cells. DNA-P was highly active in G0 + G1 cells, but maximum activity was in S cells. FH2-R exhibited maximum activity in S cells, although the difference in activity between S and G2 + M cells was not significant. None of the observed differences in MS activity was significant, indicating equally high activity in cells of all cell cycle phases. HK activity is approximately twice as high in G2 + M cells as in G0 + G1 cells.
TL;DR: In situ transcription is simpler and faster than standard methods of in situ hybridization with prelabeled cDNA or RNA probes and can be applied to the detection of any message of known sequence.
Journal Article•
TL;DR: A kinetic analysis of cell-mediated cytotoxicity (CMC) at the population level based on theoretical models has been performed, and it has been shown that these processes are governed by an equilibrium in which the unbound effector and target cells, and the conjugates formed, cannot be increased.
Abstract: A kinetic analysis of cell-mediated cytotoxicity (CMC) at the population level based on theoretical models has been performed. This analysis considers that the binding process and the kinetics of the lytic process occur through different types of conjugates: LTn, i.e., conjugates containing one effector cell and n target cells, and LmT conjugates which contain m effector cells bound to one target cell. This allowed us to provide a quantitative description of the conjugation process, and of the binding capacities of the effector and target cells. Thus, it has been shown that these processes are governed by an equilibrium in which are involved the unbound effector and target cells, and the conjugates formed. This implies that, when the equilibrium concentrations are reached, the total number of conjugates cannot be increased from the unbound effector and target cells. However, it does not mean that the free effector cells are nonbinding, and so, when the conditions of equilibrium are perturbated (as occurs for example in CMC), all effector cells, virgin and those who have already participated in the lytic process, are able to form new conjugates. The existence of this equilibrium has also important consequences when different subpopulations are separated from an effector-target system. Thus, it explains the observation reported in the literature that, although cytometric techniques can be used to detect and count different types of conjugates, the conjugates formed cannot be separated by cell sorting (unless special precautions are taken). Finally, we have found that the number of target cells killed by one effector cell, which has been previously considered as the recycling capacity of an effector population, is in reality the result of two different mechanisms. One of these mechanisms is due to the recycling process, whereas the other (which has the same effect) is due to the multiple killing capacity of the LTn conjugates which kill more than one target cell. The average number of target cells killed per conjugate has been determined, and this allowed us to obtain the relative contributions of these two mechanisms to the total killing capacity of one effector cell. It appears that the recycling capacities of effector cell populations previously determined had been 1- to 3-fold overestimated.
TL;DR: In hybrid cells, χ gene transcription was specifically turned off as demonstrated by nuclear run-on assays performed on 16-day-old proliferating hybrids, and a mechanism affecting mRNA stability may also contribute, at least initially, to the rapid depletion of cytoplasmic χ transcripts, observed during the first few hours after fusion.
TL;DR: Results indicated that P‐IgG binding is homogeneous within the living keratinocytes and increases with cell size (cell maturity), as well as with two other membrane ligands: i.e., concanavalin A and multispecific rabbit “antisurface” antibodies.
Abstract: Pemphigus is an intraepidermal autoimmune blistering disease of humans caused by circulating IgGs. We have investigated the binding mode and the fate of bound antibodies from Pemphigus sera (P-IgG) on guinea pig keratinocytes in suspension in order to find clues to the loss of cell adhesion in vivo (acantholysis). Flow cytometry, following indirect immunofluorescent labeling of the keratinocytes, and dead cells' staining with ethidium bromide, demonstrated the specific surface binding of P-IgG onto living keratinocytes only. This was shown with several Pemphigus sera or purified P-IgG. This technique, used with various Pemphigus sera, showed that the specific binding is increased when the serum titer is higher, and "Km" values for P-IgG were roughly and inversely correlated to the titers. Upon saturation the same average number of Pemphigus IgG sites per cell were found for the sera of different patients. Analysis of the specific binding of [125I]-P-IgG onto Percoll-separated (living) keratinocytes showed the existence of two classes of sites: 2 x 10(6) sites/cell high-affinity sites (Kd = 1.5 x 10(-6) M total IgG) and 25 x 10(6) sites/cell low-affinity sites (Kd = 6 x 10(-5) M total IgG). Cell sorting and flow cytometry of individual cells allowed us to correlate the light-scattering signal, the RNA content, the size and morphology, and the P-IgG binding to the cells. The results indicated that P-IgG binding is homogeneous within the living keratinocytes and increases with cell size (cell maturity). Cell-sorter analysis of cells with membrane-bound P-IgG, coupled to direct determination of P-IgG released in the medium, revealed the fate of bound P-IgG: 40-60% of the P-IgGs were released in the medium within 30 minutes at 37 degrees C. This was accompanied and followed by a much slower, metabolic energy-dependent, internalization process of the membrane-bound P-IgG. The internalization has been confirmed by electron microscopy of bound P-IgG labeled with protein A-gold. Internalized IgGs were seen in the cells in coated membranous vesicles and other endocytic compartments. Similar behavior was also observed with two other membrane ligands: i.e., concanavalin A and multispecific rabbit "antisurface" antibodies.
1 Jan 1989
TL;DR: This chapter reviews the technique of autofluorescence-activated cell sorting and discusses its usefulness in recognizing and isolating cell preparations with different functional properties, and underlines the need to purify cells according to their functional properties rather thanaccording to their structural characteristics.
Abstract: Publisher Summary This chapter reviews the technique of autofluorescence-activated cell sorting and discusses its usefulness in recognizing and isolating cell preparations with different functional properties. Earlier studies demonstrated how the measurement of the cellular FAD and NAD(P)H fluorescent intensities allows the distinction and isolation of various types of pancreatic endocrine cells. Autofluorescence-activated cell sorting permitted the preparation of pure and single insulin-containing B cells, which were used to investigate the functional properties of this cell type without interference from other cell types. In the course of these studies, it was noticed that this endocrine cell population does not represent a homogeneous group of identical cells, but rather is composed of functionally diverse subpopulations. Several of these subpopulations have been recognized during flow cytometric analysis, whereby the cellular autofluorescent compounds FAD and NAD(P)H appeared again to be particularly powerful discriminators. The observation that a pure pancreatic B-cell population is composed of functionally diverse subpopulations underlines the need to purify cells according to their functional properties rather than according to their structural characteristics.
TL;DR: Although cells derived from the bone marrow or peripheral blood of twenty patients with myeloproliferative disorders, myelodysplastic syndromes or acute myeloid leukemia frequently showed proliferative responses to IL3, mRNA transcripts for IL3 were not detected in these cells, implying that autocrine secretion of IL3 was not the mechanism by which these leukemias were maintained.
Abstract: The effects of recombinant human interleukin 3 (IL3) on normal bone marrow cells and human leukemic cells were studied. In clonal assays, IL3 supported the growth of all colony types including megakaryocytes. Erythroid colonies were formed in the presence of IL3 and erythropoietin, but not in the absence of erythropoietin. Replating experiments using blast cell colonies derived from a cell population enriched for progenitor cells by fluorescence-activated cell sorting with the monoclonal antibody 3C5, showed that IL3 supported the continued replating of colonies. The clonal proliferation of human bone marrow cells in response to IL3 was inhibited by tumor necrosis factor and by lymphotoxin, but not by interferon-γ. In suspension cultures, IL3 supported the proliferation of mast cells. Human IL3 had no effect on the growth responses, morphology, cytochemistry, or clonogenicity of the human leukemic cell lines HL60, U-937, KG1a, and HEL. Transcripts for IL3 mRNA were not detectable in these cells, n...
TL;DR: The present report describes the analysis and cell sorting of a population of retrogradely fluorescence-labeled dopamine-containing neurons from ventral mesencephalon that differentiates toward a mature phenotype bearing a prominent neuropil.
TL;DR: It is concluded that ras oncogene expression in these cells can influence the induction of class II antigen but that because ras expression in the sorted lines is similar the effect is indirect and presumably involves interaction with other cellular factors.
TL;DR: FCM appears to be useful as a new tool for sensitive detection of transient expression of heterologous reporter genes in COS-7 cells for protoplast fusion and transfer efficiencies.
Abstract: Transfer and expression of a plasmid containing the gene encoding the human T-cell antigen CD4 by protoplast fusion was measured by flow cytometry (FCM). Protoplasts were prelabeled with fluorescein isothiocyanate (FITC) and fused to COS-7 cells. Nonspecific protoplast adsorption to the plasma membrane was differentiated from successful protoplast fusion by the addition of an antibody directed against fluorescein to quench extracellular protoplast fluorescence. Transfection efficiencies were defined as both percent CD4 expressing cells and CD4 expression levels on a single cell basis in the transient immunofluorescence assay. Cell sorting studies indicated that intracellular protoplast-associated fluorescence immediately after fusion exhibited a good correlation with transient CD4 transfection efficiencies as measured by indirect immunofluorescence. Reconstruction experiments comparing CD4 transfer efficiencies of protoplast fusion and calcium phosphate transfection showed that fusion resulted in a higher percentage of CD4 expressing transfectants, while calcium phosphate transfection yielded higher CD4 expression levels on a single cell basis. Thus, FCM appears to be useful as a new tool for sensitive detection of transient expression of heterologous reporter genes in COS-7 cells.
TL;DR: Monoclonal antibodies suggest that the interaciton of T‐sRFC with SRBC occurs at or near the T cell receptor, and it is shown that the vast majority of spleen sRFC have the L3T4−Ly‐2+ phenotype.
Abstract: Spontaneous sheep erythrocyte rosette-forming T cells (T-sRFC) found in normal mouse spleen are specific for the xenogeneic species of red cells and are distinct from human E rosettes as illustrated by their persistence after sheep red blood cell (SRBC) incubation with anti-LFA-3 monoclonal antibodies directed against SRBC molecule binding to the E rosette receptor. We confirm here using monoclonal antibodies that T-sRFC are Thy-1+CD3+. Additionally, using cell separation techniques based on panning and cell sorting, it is shown that the vast majority of spleen sRFC have the L3T4−Ly-2+ phenotype. As already shown for anti-Thy-1 antibodies, anti-CD3 and anti-Ly-2 antibodies block rosette formation, whereas antibodies to more abundant cell surface antigens (T200, H-2Db) are not inhibitory. These data suggest that the interaciton of T-sRFC with SRBC occurs at or near the T cell receptor.