An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide

A rapid, simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is described for use in the determination of cell viability in cell suspension. Air-dried slide preparations can be made from the cell suspensions so that an accurate estimate of the viability of the cells in the original suspension can be made up to 1 week later. Viable cells fluoresce bright green, while nonviable cells are bright red. Furthermore, when FDA-PI staining is compared to trypan blue dye exclusion as a method to determine cell viability, FDA-PI is found to be more consistent over prolonged periods of exposure to the dyes. Therefore, double staining with FDA-PI is a rapid, convenient, and reliable method to determine cell viability.     

Rapid assessment of islet viability with acridine orange and propidium iodide

Summary A simple, rapid method for estimating the viability of isolated islets of Langerhans with fluorescent dyes is described. Low concentrations of acridine orange and propidium iodide (AO/PI) were used to visualize living and dead islet cells simultaneously. AO/PI-stained islets can be divided into three distinct groups. Group A islets fluoresce green, contain insulin, and have normal ultrastructure; group C islets fluoresce primarily red, contain little or no insulin, and have cells with disrupted cellular membranes. Group B islets fluoresce red, green, and yellow. The yellow color is due to the addition of two primary colors from the superimposed red and green fluorescing cells. In this assay, the interpretation that red islet cells are dead and green islet cells are alive was confirmed by sequentially staining single islet cells with AO/PI and trypan blue. The observation that red islets are dead was confirmed by heat-killing, enzymatically damaging, treating with ethanol, or depriving islets of nutrients and observing the red fluorescence. This assay should be useful in studies where the assessment of islet viability is essential. Preliminary reports of this work were presented at two meetings and were published in abstract form (24,25). This research was supported in part by the National Institutes of Health, Bethesda, MD, grant DK 18115.     

Changes in distribution of nuclear matrix antigens during the mitotic cell cycle

We examined the distribution of nonlamin nuclear matrix antigens during the mitotic cell cycle in mouse 3T3 fibroblasts. Four monoclonal antibodies produced against isolated nuclear matrices were used to characterize antigens by the immunoblotting of isolated nuclear matrix preparations, and were used to localize the antigens by indirect immunofluorescence. For comparison, lamins and histones were localized using human autoimmune antibodies. At interphase, the monoclonal antibodies recognized non-nucleolar and nonheterochromatin nuclear components. Antibody P1 stained the nuclear periphery homogeneously, with some small invaginations toward the interior of the nucleus. Antibody I1 detected an antigen distributed as fine granules throughout the nuclear interior. Monoclonals PI1 and PI2 stained both the nuclear periphery and interior, with some characteristic differences. During mitosis, P1 and I1 were chromosome-associated, whereas PI1 and PI2 dispersed in the cytoplasm. Antibody P1 heavily stained the periphery of the chromosome mass, and we suggest that the antigen may play a role in maintaining interphase and mitotic chromosome order. With antibody I1, bright granules were distributed along the chromosomes and there was also some diffuse internal staining. The antigen to I1 may be involved in chromatin/chromosome higher-order organization throughout the cell cycle. Antibodies PI1 and PI2 were redistributed independently during prophase, and dispersed into the cytoplasm during prometaphase. Antibody PI2 also detected antigen associated with the spindle poles.     

The role and clinical implications of G6PI in experimental models of rheumatoid arthritis

The antigens that trigger the pathogenic immune response in rheumatoid arthritis (RA) remain unknown. Until recently it was assumed that either viral or microbial antigens, or joint-specific antigens were the target of arthritogenic T and B lymphocytes in RA. Consequently, murine models of arthritis are induced by immunization with either joint-specific antigens such as type II collagen or microbial products such as streptococcal cell wall. In the K/BxN T-cell receptor transgenic mouse model arthritis is caused by a systemic autoimmune response to the ubiquitously expressed glycolytic enzyme glucose-6-phosphate isomerase (G6PI). The autoreactive transgenic T cells recognize G6PI and provide help for the production of arthritogenic IgG antibodies against G6PI. More recently it was shown that G6PI immunization induces severe symmetrical peripheral polyarthritis in genetically unaltered DBA/I mice. In that model CD4+ T cells are necessary not only for the induction but also for the effector phase of arthritis. Here we review the pathomechanisms that lead from systemic autoreactivity to arthritis in these models, consider the relevance of anti-G6PI immune reactivity for RA, and discuss the insights into the pathogenesis of RA and possibly other autoimmune conditions that can be gained from these models.     

The role and clinical implications of G6PI in experimental models of rheumatoid arthritis

The antigens that trigger the pathogenic immune response in rheumatoid arthritis (RA) remain unknown. Until recently it was assumed that either viral or microbial antigens, or joint-specific antigens were the target of arthritogenic T and B lymphocytes in RA. Consequently, murine models of arthritis are induced by immunization with either joint-specific antigens such as type II collagen or microbial products such as streptococcal cell wall. In the K/B×N T-cell receptor transgenic mouse model arthritis is caused by a systemic autoimmune response to the ubiquitously expressed glycolytic enzyme glucose-6-phosphate isomerase (G6PI). The autoreactive transgenic T cells recognize G6PI and provide help for the production of arthritogenic IgG antibodies against G6PI. More recently it was shown that G6PI immunization induces severe symmetrical peripheral polyarthritis in genetically unaltered DBA/I mice. In that model CD4⁺ T cells are necessary not only for the induction but also for the effector phase of arthritis. Here we review the pathomechanisms that lead from systemic autoreactivity to arthritis in these models, consider the relevance of anti-G6PI immune reactivity for RA, and discuss the insights into the pathogenesis of RA and possibly other autoimmune conditions that can be gained from these models.     

Cell-Cycle Dependent Appearance of Murine Leukemia-Sarcoma Virus Antigens

Normal rat kidney (NRK) cells, NRK cells infected with Rauscher murine leukemia virus, and NRK cells infected with Kirsten murine sarcoma-leukemia virus (NRK-K) were synchronized by a double thymidine block. At intervals after release from thymidine blockage, the cells were examined for the presence of viral antigens in the cytoplasm and on the cell surface by immunofluorescent microscopy by using goat anti-Rauscher murine leukemia virus and goat anti-Moloney leukemia virus (Tween-ether disrupted) sera. Detection of viral antigens in the cytoplasm was periodic during the cell cycle. Antigens were detected first during the S phase, increased during the G2 phase, and disappeared during the M and G1 phases. A similar pattern of surface immunofluorescence was observed. Infectious virus was detected in culture fluids from synchronized cells during the M phase. Surface immunofluorescence was detected in NRK-K cells with anti-Rauscher murine leukemia virus and may represent the presence of group-specific antigens on the cell surface. Control, uninfected NRK cells, which did not normally fluoresce, showed weak immunofluorescence during the S and G2 phases after synchronization. Synchronization can be used to amplify latent oncornavirus expression.     

Growth Kinetics of Yaba Tumor Poxvirus After In Vitro Adaptation to Cercopithecus Kidney Cells

Yaba tumor poxvirus has been adapted to continuous in vitro cultivation in monolayers of cercopithecus kidney cells. At 35 C, the minimum replicative cycle, after synchronous infection of CV-1 cells with multiplicity of infection of 135 focusforming units per cell, was 35 hr; however, maximum virus yields were not obtained until 75 hr postinfection (PI). Cytoplasmic incorporation of (3)H-thymidine [viral deoxyribonucleic acid (DNA) synthesis] was detected 3 hr PI and was preceded by synthesis of nonstructural associated antigens (YS). Synthesis of YS antigens was not inhibited by the DNA inhibitor, arabinofuranosyl cytosine (ARA-C). Synthesis of at least two virion structural antigens, although not detected by immunofluorescence until 2 hr after the onset of DNA synthesis, occurred in the presence of ARA-C, indicating potential translation of these structural antigens from parental DNA. The first progeny DNA was completed by 20 hr PI but was not detected in infectious form until 35 hr PI. The maximum rate of progeny DNA completion occurred between 20 and 30 hr PI. DNA synthesis continued 45 to 50 hr PI. The adapted virus retained its oncogenicity and, like the wild type, replicated better at 35 C than at 37 C. A synthetic step associated with viral DNA synthesis appears to be temperature-sensitive.     

The effect of inoculum concentration on the development of the nuclear polyhedrosis virus of Autographa californica in TN-368 cells

A multiplicity of infection (m.o.i.) of 25 or 50 mean tissue culture-infective doses (TCID₅₀) of Autographa californica NPV per cell of a TN-368 cell line initially infected >90% of attached cells whereas an m.o.i. of 1 or 5 TCID₅₀/cell initially infected <50% of the cells. An immunoperoxidase technique first detected nucleocapsid antigens at 6–12 hr postinfection (PI) and polyhedral protein antigen 12–18 hr PI, which was followed 4–6 hr later by polyhedra formation. At a m.o.i. of 50, the extracellular virus titer (nonoccluded progeny virus) increased between 6 and 12 hr PI while at m.o.i. of 25, 5, and 1, the titer increased at 12–18 hr PI. Antisera to nucleocapsids and polyhedral protein were specific and also failed to react with viral envelope antigens.     

Binding of phosphatidylethanolamine and phosphatidylinositol to OKT8+ lymphocytes activates suppressor cell activity

The phospholipids phosphatidylethanolamine (PE) and phosphatidylinositol (PI) have been shown to activate a population of OKT8-enriched lymphocytes to become activated suppressor cells that result in the suppression of lymphocyte blastogenesis to a variety of mitogens and antigens. This suppression is dose dependent, and maximal suppressor activity is obtained at concentrations of 125 micrograms/ml PE and 25 micrograms/ml PI. Activation of the suppressor cell population is not associated with an actual increase in the number of cells expressing the OKT8 antigen, but the number of these cells expressing Dr antigens on their surface was increased. Both PE and PI bound to lymphocytes in a specific manner. Binding of radiolabeled PE could be inhibited by unlabeled PE but not by PI or phosphatidylserine (PS). Similarly, the binding of PI to lymphocytes was also found to be specific. Although radiolabeled PE bound to lymphocytes other than OKT8+ cells, and to other peripheral leukocytes, it bound to OKT8+ cells with a significantly greater affinity than it did to the other cell types. The Kd for PE was 1 x 10(2) nM and for PI was 1 x 10(3) nM, and receptor cell densities for these two phospholipids were estimated at 1 x 10(-8) nM and 3 x 10(-9) nM, respectively. The receptors for these two phospholipids were trypsin and heat sensitive, and the receptor sites could be regenerated after a 24-hr incubation after trypsinization.     

The phosphatidylinositol response is an early event in the physiologically relevant activation of antigen-specific B lymphocytes

Receptor ligand-induced turnover of plasma membrane phosphatidylinositol (PI) has been implicated as part of a membrane receptor signal transduction system in a number of mammalian cell types. Signaling through B-lymphocyte surface immunoglobulin (sIg2) has been explored polyclonally through the use of anti-Ig reagents, with the assumption that anti-Ig mimics the process of antigen binding to the antigen-specific cell. We have utilized a method of obtaining trinitrophenyl (TNP)-specific populations of B lymphocytes in order to determine if antigen binding to these antigen-specific cells initiates PI turnover. This method has allowed us to explore the membrane phospholipid events following antigen binding directly, rather than with inference from the anti-Ig system. We have found that both thymus-dependent and thymus-independent antigens (with the exception of TNP-lipopolysaccharide) produced an increase in PI turnover comparable to that generated by anti-IgM stimulation. The lack of increased PI turnover following TNP-LPS stimulation may be attributable to the action of LPS on the biochemical events of the PI cycle. In a B-cell subpopulation depleted of antigen-specific cells, only anti-IgM produced a PI effect. These results represent the first demonstration of PI turnover as an early activation event in a physiologically relevant lymphocyte system.     

Antigenemia and Specific IgM and IgG Antibody Responses in Rabbits Infected with Toxoplasma gondii

In this experiment, the correlation between antigenemia and specific antibody responses in Toxoplasma gondii-infected rabbits was assessed. We injected 1,000 T. gondii tachyzoites (RH) subcutaneously into 5 rabbits. Parasitemia, circulating antigens, and IgM and IgG antibody titers in blood were tested by ELISA and immunoblot. For detection of parasitemia, mice were injected with blood from rabbits infected with T. gondii and mice died between days 2 and 10 post-infection (PI). Circulating antigens were detected early on day 2 PI, and the titers increased from day 4 PI and peaked on day 12 PI. Anti-Toxoplasma IgM antibody titers increased on day 6 PI and peaked on days 14-16 PI. IgG was detected from day 10 PI, and the titers increased continuously during the experiment. The antigenic protein patterns differed during the infection period, and the number of bands increased with ongoing infection by the immunoblot analysis. These result indicated that Toxoplasma circulating antigens during acute toxoplasmosis are closely related to the presence of parasites in blood. Also, the circulating antigen levels were closely correlated with IgM titers, but not with IgG titers. Therefore, co-detection of circulating antigens with IgM antibodies may improve the reliability of the diagnosis of acute toxoplasmosis.     

Correlated analysis of cellular DNA, membrane antigens and light scatter of human lymphoid cells

Flow cytometric correlated analysis of membrane antigens, DNA, and light scatter was performed on human lymphoid cells using fluorescein (FITC)-conjugated antibodies to label B- and T-cell antigens and propidium iodide (PI) to stain DNA after ethanol fixation and RNase treatment. A FACS II flow cytometer was modified to obtain digitized measurements of two color fluorescence and light scatter emissions, simultaneously. Software was written to allow single parameter analysis or correlated analysis of any two of the three parameters acquired. Ethanol fixation preserved FITC surface labeling for at least 15 weeks, but produced marked changes in light scatter. No changes in FITC distributions were observed after RNase treatment and PI staining, and the presence of FITC labeling did not affect DNA distributions. Within heterogeneous cell populations, the DNA distribution of cell subpopulations identified by a membrane antigen was clearly demonstrated.     

Membrane cell permeabilization with saponin and multiparametric analysis by flow cytometry.

Saponin, a detergent like molecule, can permeabilize cell membranes without destroying them, and thus can be used for the detection of intracellular antigens on intact cells with a flow cytometer. First experiments were reported that demonstrated the detection of intracytoplasmic antigens such as intermediate filaments and CD3 in T acute lymphoblastic leukemia (ALL). Further experiments were also performed to prove that intranuclear structures were equally accessible: dyes such as propidium iodide (PI) and monoclonal antibodies (mAb) such as Ki67 could penetrate the nucleus and lead to the analysis of DNA content and to the discrimination between the different cell cycle phases (G0, G1, S, G2-M). This rapid and sensitive method retained sufficient integrity of cells being treated to enable differentiation of cell types on the basis of morphology. Furthermore, it did not alter membrane expression of most antigens. Therefore, it was of particular interest for multiparametric analysis, especially for simultaneous study of membrane and intracellular structures.     

Temporary competitive inhibition of a tumour cell surface protease as a protective mechanism in the preparation of the membrane bound native enzyme in the presence of excess cytoplasmic inhibitors.

Tumour cells possess a cell surface protease which is recognised and inhibited by a cytoplasmic protein extractable from frozen sections of tumour cells. In order to prepare sections with tumour cells carrying cell surface-bound native protease in the absence of this internal inhibitor we have used a reversible competitive inhibition step as a temporary measure to protect the active centre of GB whilst the cytoplasmic inhibitor is extracted from the frozen sections. These sections are described as protected in the sense that the enzyme is native and fully functional now that potential inhibitors have been extracted. The protected cell surface protease immobilised in the cell surface of squamous cell carcinoma cells has been used as the target for inhibition studies and displacement studies. The ability to follow these inhibition and exchange reactions concerning the cell surface protease has been made possible by virtue of the fluorescent probe, 9-amino acridine, which locates the active centre of the protease. Cells with active protease bind 9-amino acridine and fluoresce yellow; cells lacking this protease or having inhibited protease fail to bind 9-amino acridine and do not fluoresce.     

Quantifying the syncytialisation of human placental trophoblast BeWo cells grown in vitro

We have generated lines of BeWo cells that constitutively and stably express either histone H2B tagged with the green fluorescent protein (GFP), or the mitochondrial targeting sequence of subunit VIII of cytochrome c oxidase fused with a red fluorescent protein; one line has nuclei that fluoresce green, the other mitochondria that fluoresce red. Expression of these tagged proteins has no effect on the rates of DNA, RNA and protein synthesis, or on the amounts of human chorionic gonadotropin (hCG) secreted after treatment with forskolin. We used fluorescence-activated cell sorting (FACS) to monitor the extent of cell fusion (syncytialisation) between these two lines; fused cells are readily and accurately detected by their green/red fluorescence. This assay should prove useful in the investigation of the molecular mechanisms involved in trophoblast syncytialisation.     

Modulation of lymphocyte nuclear matrix organization in vivo by 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole: an autoradiographic and immunofluorescence study

Assembly of active nuclei in lymphocytes stimulated by mitogen is paralleled by the elaboration of a structurally and biochemically complex nuclear matrix (NM). To examine the dynamics of individual NM polypeptide components during blastogenesis, we have applied immunofluorescence labelling with anti-NM antibodies to concanavalin A-stimulated mouse splenocytes. Whereas peripherin and PI2 antigens did not reorganize during stimulation, labelling of PI1 and small nuclear ribonucleoprotein (snRNP) antigens increased markedly in intensity and redistributed in concert with the previously reported NM restructuring. Double-labelling showed, furthermore, that snRNPs and the internal staining component of PI1 were largely co-localized. As an approach to studying the role of RNA and RNA synthesis in NM organization, we have further examined the effects of the inhibitor of RNA synthesis, 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), on NM antigen distribution. The rapid inhibition of 3H-uridine incorporation by DRB was accompanied by coordinate aggregation of snRNPs and of the internal PI1 component into large, brightly stained patches. Both 3H-uridine incorporation levels and antigen localization were readily reversed upon removal of DRB. We conclude that NM antigens behave independently during nuclear and NM assembly and that NM organization, as reflected by NM antigen distribution, is modulated by con A- and DRB-induced alterations in RNA synthesis. We propose, furthermore, that the PI1 antigen plays a role in RNA metabolism, and is possibly involved in RNA transport to the nuclear periphery.     

Antigenic profile and localization of Clonorchis sinensis proteins in the course of infection.

In the course of Clonorchis sinensis infection, antigens presented to the hosts may be in a close relation to growth of the fluke. The antigenic proteins stimulating IgG antibody production were chronologically identified by immunoblot and localized by immunohistochemical staining. In the early stage of infection until 12 weeks post-infection (PI), antigens were proteins with molecular mass larger than 34 kDa which were derived from the tegument, testes and intrauterine eggs. After 20 weeks PI, antigens recognized were 29, 27 and 26 kDa proteins from the intestine, excretory bladder and reproductive organs. It is suggested that the tegumental proteins are the most potent antigens and the excretory-secretory proteins with middle molecular mass of 26-45 kDa contribute to the high level production of antibodies after 20 weeks of the C. sinensis infection.     

A role for phosphatidylinositol 3-kinase in the regulation of beta 1 integrin activity by the CD2 antigen

The rapid and reversible upregulation of the functional activity of integrin receptors on T lymphocytes is a vital step in the adhesive interactions that occur during successful T cell recognition of foreign antigen and transendothelial migration. Although the ligation of several different cell surface receptors, including the antigen- specific CD3/T cell receptor complex, the CD2, CD7, and CD28 antigens, as well as several chemokine receptors, has been shown to rapidly upregulate integrin function, the intracellular signaling events that initiate this increase in adhesion remain poorly defined. In this study, we have used DNA-mediated gene transfer to explore the role of phosphatidylinositol 3-kinase (PI 3-K) in the upregulation of beta 1 integrin functional activity mediated by the CD2 antigen. CD2 was expressed in the myelomonocytic cell line HL60, which expresses beta 1 integrins that mediate adhesion to fibronectin and VCAM-1 in an activation-dependent manner. Antibody stimulation of CD2 expressed on HL60 transfectants resulted within minutes in increased beta 1-mediated adhesion to fibronectin and VCAM-1 at levels comparable to that obtained upon stimulation with the phorbol ester PMA. A role for PI 3-K in CD2-mediated increases in beta 1 integrin function is suggested by: (a) the ability of the PI 3-K inhibitor wortmannin to completely inhibit CD2-induced increases in beta 1 integrin activity; (b) the association of PI 3-K with CD2; and (c) induced PI 3-K activity upon CD2 stimulation. The mode of association of PI 3-K with CD2 is not mediated by tyrosine phosphorylation-dependent binding of PI 3-K via SH2 domains, since: (a) PI 3-K is associated with CD2 in unstimulated cells; (b) CD2 stimulation fails to increase the amount of associated PI 3-K; and (c) the CD2 cytoplasmic domain lacks tyrosine residues. A role for both protein kinase C and cytoskeletal rearrangements in CD2 regulation of integrin activity is also suggested, since a PKC inhibitor partially inhibits CD2-induced increases in beta 1 integrin function, and CD2 stimulation increases F-actin content in a wortmannin- sensitive manner. Analysis of human peripheral T cells indicated that CD2 stimulation also results in PI 3-K-dependent upregulation of beta 1 integrin activity. Thus, these results demonstrate that CD2 can function as an adhesion regulator in the absence of expression of the CD3/T cell receptor complex; and directly implicate PI 3-K as a critical intracellular mediator involved in the regulation of beta 1 integrin functional activity by the CD2 antigen.     

Stage-specific antigens of Nematospiroides dubius Baylis, 1926 (Nematoda: Heligmosomides)

Antigens were identified from Nematospiroides dubius recovered from outbred Quackenbush mice between 4 and 10 days postinoculation (PI). Parasite surface proteins were radioiodinated and extracts of the whole worms were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and reacted with normal and immune mouse sera followed by an avidin-biotin-peroxidase assay. Antigens ranged between 250,000 and 20,000 molecular weight (MW). A major surface antigen, 60,000 MW, which appeared to be a complex of different antigens, and a 250,000-MW internal antigen were found on fourth-stage (L4) and fifth-stage (L5) larvae 5-10 days PI but not earlier. A group of minor surface antigens (24,000-30,000 MW) were also expressed as larvae molted from L4 to L5, 6 and 7 days PI, but they differed from antigens of similar MW expressed by adult worms. An antigen, 45,000 MW, was detected in worms 5-10 days PI, but it was only expressed on the surface of L5 worms 9 and 10 days PI. We suggest that the antigen(s) common to adults and larvae may account for protective immunity.     

A Novel Technique for Viable Cell Determinations

We have developed a simple method to determine cell viability using two fluorescent dyes, Hoechst 33258 and acridine orange. When these dyes are used in combination, dead cells fluoresce brilliant blue and live cells fluoresce green. This method works over a range of dye concentrations (Hoechst 33258, 0.25-2 μg/ml; acridine orange, 1-5.0 μg/ml) and the fluorescence spectra of the two dyes are such that only one set of filters is required to visualize the effects of both dyes simultaneously. It is insensitive to a wide range of exogenous serum concentrations and is read with greater uniformity by different observers.