DAPI as a Useful Stain for Nuclear Quantitation

A simple-to-use fluorescent stain, 4′,6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA in both living and fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI. DAPI staining allows multiple use of cells eliminating the need for duplicate samples.     

DAPI as a Useful Stain for Nuclear Quantitation

A simple-to-use fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA in both living and fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI. DAPI staining allows multiple use of cells eliminating the need for duplicate samples.     

The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem

Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4′,6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detected with blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. We also suggest avoiding the use of mounting medium with high glycerol concentrations since glycerol showed the strongest impact on photoconversion. This photoconversion effect cannot be avoided even when using narrow bandpass filter sets.     

Screening of murine monoclonal antibodies against living schistosomula of Schistosoma mansoni by radioimmunoassay

A radioimmunoassay was developed to screen supernatants of murine monoclonal antibodies against surface antigens of living schistosomula of Schistosoma mansoni. Of 196 clones screened, 10% bound schistosomula. Of these, 74% bound only schistosomula. The remaining molecules also reacted with soluble adult worm antigens and soluble egg antigens as determined by enzyme-linked immunosorbent assay. Immunoblot analysis demonstrated that monoclonal antibody 204-3E4 reacted with a 68 kDa protein, a glycoprotein that induces substantial resistance against S. mansoni infection. Recognition of an 18 kDa antigen by 204-3F1 antibody was stage-specific with the antigen being expressed in cercariae, 3- and 24-h-old parasites but not 4-day, lung stage or adult worms. Monoclonal antibody 204-4E3 reacted with purified S. mansoni paramyosin. These data indicate that radioimmunoassay using living schistosomula is a rapid alternative method to identify murine hybridomas that secrete antibodies which react with surface antigens of S. mansoni.     

Macrophages as effector cells of protective immunity in murine schistosomiasis. VI. T cell-dependent, lymphokine-mediated, activation of macrophages in response to Schistosoma mansoni antigens

Macrophages from Schistosoma mansoni-infected mice kill significant numbers of skin stage schistosomula and murine fibrosarcoma cells in vitro. In order to determine whether the macrophage tumoricidal and larvicidal activation observed in mice as a result of S. mansoni infection are mediated through T cell-dependent (lymphokine) or B cell-dependent (antibody or immune complex) mechanisms, the development of macrophage populations with cytotoxic activity against schistosome larvae or tumor cells was monitored in S. mansoni-infected nude or mu-suppressed mice. Whereas peritoneal cells from S. mansoni-infected congenitally athymic mice had no activity in either assay, cells from mu-suppressed S. mansoni-infected mice showed cytotoxic activity equivalent to that of cells from untreated S. mansoni-infected counterparts. Cells from mu-suppressed uninfected mice were not activated. The mu-suppressed animals had no detectable nonspecific IgM or specific antischistosome IgM, IgG, or IgE antibodies and showed a 90% reduction in numbers of splenic IgM+ cells upon fluorescence activated cell sorter analysis. These results indicate that antibody is not required for in vivo activation of macrophages during S. mansoni infection. Further experiments showed that lymphoid cells from S. mansoni infected mice respond in culture with various specific antigens (such as living or dead whole schistosomula or soluble adult worm antigens) by production of factors capable of activating macrophages from uninfected control mice to kill schistosomula or tumor cells in vitro. Macrophage-activating factors were produced by T cell-enriched, but not T cell-depleted or B cell-enriched, populations from spleens of schistosome-infected mice in response to schistosome antigen. Similar lymphokines may be responsible for the macrophage activation observed during chronic murine schistosomiasis. These observations emphasize the potential contribution of T cell-mediated immune mechanisms in resistance to S. mansoni infection.     

Different effects of 33258 Hoechst and DAPI in fluorescent staining of sister chromatids differentially substituted with bromodeoxyuridine

Summary After substitution with 5-bromodeoxyuridine (BrdUrd) for two rounds of replication, chromosomes in cytological preparations stained with 33258 Hoechst show upon epiluminescence an immediate differential sister chromatid fluorescence. When stained with DAPI, however, which has a structural resemblance to part of the 33258 Hoechst molecule, such a differential pattern of fluorescence was only induced after some delay. Upon restaining with the same dye the differential fluorescence appeared instantly. In preparations double stained with ethidium bromide and 33258 Hoechst the induction of a differential staining of sister chromatids with 33258 Hoechst was not accompanied by a differential staining with ethidium bromide. Once a differential staining was obtained with DAPI in preparations double stained with ethidium bromide and DAPI, the ethidium bromide pattern also appeared to be differential upon subsequent observation. No differentiation could be obtained with ethidium bromide alone. The observations described in the case of 33258 Hoechst staining are in agreement with a molecular quenching by BrdUrd without gross structural consequences for the DNA. In the case of DAPI staining, however, there occurs a differential photolysis of BrdUrd-substituted DNA. Besides the nature, most likely the size, of the fluorochrome molecules themselves, the state of the fixed chromatin appeared also to play a role in determining the mechanism of the sister chromatid differentiation: after prolonged incubation in buffer, BrdUrd-containing chromosomes stained with 33258 Hoechst showed a differential staining evidently caused by photolysis, indicating that they had become more susceptible to light.     

"Liquidless" cell staining by dye diffusion from gels and analysis by laser scanning cytometry: potential application at microgravity conditions in space

BACKGROUND: Conventional staining of cells or tissue sections on microscope slides involves immersing the slides into solutions of dyes then rinsing to remove the unbound dye. There are instances, however, when use of stain solutions is undesirable-e.g., at microgravity conditions in space, where the possibility of accidental spill (many dyes are known carcinogens) introduces health hazard. Likewise, transporting bulk of liquid stains and rinses may be burdensome in certain situations such as field expeditions or combat. METHODS: The "liquidless" staining procedure is proposed in which the dyes are contained in thin strips of hydrated polyacrylamide or gelatin gels that have been presoaked in the stain solutions. Fluorochromes that have affinity to DNA (propidium iodide, PI; 4,6-diamidino-2-phenylindole, DAPI, Hoechst 33342) or to protein (sulforhodamine 101) were used to saturate the gels. The gel strips were placed over the prefixed cells or tissue sections deposited on microscope slides and relatively low (20 g/cm2) pressure was applied to ensure the contact. The cells were also stained by using commercially available mounting media into which DAPI or PI were admixed. Intensity of fluorescence of the PI stained cells was measured by laser scanning cytometry (LSC). RESULTS: Satisfactory cell and tissue staining, with minimal background, was achieved after 10-20 min contact between the cells and gels. Optimal concentrations of the dyes in the solutions used to presoak the gels was found to be 2-4-fold higher than the concentrations used routinely in cytometry. The measurements of intensity of cellular fluorescence by LSC revealed that the staining of DNA was stoichiometric as reflected by the characteristic cellular DNA content frequency histograms with distinct G1, S, and G2/M cell populations and 2:1 ratio of G2/M to G1 peak fluorescence. Individual gels can be saturated with more than a single dye-e.g., to obtain differential DNA and protein staining. Cell staining with DAPI or PI in the gelatin-based mounting media led to high fluorescence background while staining with DAPI in "aqueous" medium was satisfactory. CONCLUSIONS: Relatively fast staining of cells or tissue sections on microscope slides can be achieved by nonconvective dye diffusion using hydrated gels permeated with the dyes, applied to cells at low pressure. The quality of the staining provided by this methodology is comparable to conventional cell staining in dye solutions.     

Caffeine dissociates complexes between DNA and intercalating dyes: application for bleaching fluorochrome-stained cells for their subsequent restaining and analysis by laser scanning cytometry

BACKGROUND: Removal of the nucleic acid-bound fluorochrome is desirable when stained cells have to be reanalyzed using other fluorochromes. It is also often desirable to remove DNA-bound antitumor drugs from drug-treated cells, to improve cell staining. We have previously observed that in aqueous solutions, the methylxanthine caffeine (CFN) decreases interactions between planar aromatic molecules such as intercalating dyes or antitumor drugs and nucleic acids. The aim of this study was to explore whether this property of CFN can be utilized to remove the DNA-bound intercalating dyes propidium iodide (PI) or 7-aminoactinomycin D (7-AAD) from the cells and whether the bleached cells can be restained and reanalyzed. METHODS: HL-60 cells were fixed in 70% ethanol and their DNA was stained with PI or 7-AAD. The cells were then rinsed with a 0.05 M solution of CFN in phosphate-buffered saline (PBS) or with PBS alone. The decrease in intensity of cell fluorescence during rinsing was measured by laser scanning cytometry (LSC) to obtain the bleaching kinetics of individual cells. The bleached cells were then restained with PI, 7-AAD, or the protein-specific fluorochrome sulforhodamine 101(S101). Their fluorescence was measured again by LSC. In addition, free DNA was subjected to gel electrophoresis, DNA bands in the gels were stained with ethidium bromide (EB), and the gels were rinsed with a solution of CFN or PBS to bleach the DNA band's fluorescence. RESULTS: Rinsing the PI or 7-AAD-stained cells with solutions of CFN led to nearly complete removal of PI and a more than 75% decrease in 7-AAD fluorescence after 10 min. The rinse with PBS decreased the PI cell fluorescence intensity by less than 30% and the 7-AAD fluorescence by about 50%. The differences in kinetics of PI or 7-AAD removal by CFN from G2/M versus G1 cells suggest that these intercalators bind more strongly to DNA in chromatin of G2/M than G1 cells. The CFN-bleached cells were then successfully stained with S101 and again with PI or 7-AAD. The bivariate analysis of the LSC merged files of the cells sequentially stained with PI and S101 revealed typical DNA/protein distributions. The fluorescence of EB-stained DNA bands in gels was also nearly completely removed by rinsing gels in 0.05 M CFN; PBS alone had a distinctly lesser effect. CONCLUSION: Solutions of CFN can dissociate the DNA-bound PI, 7-AAD, EB, and possibly other intercalating fluorochromes. The bleached cells can be restained and reanalyzed by LSC. This approach can also be used to remove such fluorochromes from nucleic acids immobilized in gels and perhaps in other solid matrices. Analysis of the kinetics of fluorochrome removal from cells can possibly be used to study their binding affinities to nucleic acids in situ.     

DAPI staining improved for quantitative cytofluorometry

DNA-DAPI complexes emit strong bluish white fluorescence when excited by ultraviolet light so that even very small amounts of DNA such as those in mitochondria, chloroplasts, and virus particles can be visualized. Moreover, the staining procedure with DAPI is very simple and requires no hydrolysis. However, DAPI staining was considered unsuitable for quantitative purpose; nonspecific cytoplasmic fluorescence, scattering of strong emission light, and fading of the fluorescence under UV excitation were major problems of DAPI staining in quantitative cytofluorometry. We found that (1) nonspecific cytoplasmic fluorescence could be eliminated by reducing the DAPI concentration to 50 ng/ml, (2) fluorescence decay was markedly decreased by adding electron donors and molecules containing SH radicals in the mounting media, and (3) light scattering became negligible after reducing the intensity of the excitation light. Thus satisfactory precision could be obtained in DNA quantification by epifluorescent cytophotometry on DAPI stained specimens.     

Fluorescence image cytometry of nuclear DNA content versus chromatin pattern: a comparative study of ten fluorochromes.

This study is intended to be the first step of an in situ exploration of the intranuclear DNA distribution by image cytometry (SAMBA) with several fluorochromes. The nuclear DNA content and the chromatin pattern, revealed by ten fluorochromes (HO, DAPI, MA, CMA3, OM, QM, AO, EB, PI, and 7-AMD), were analyzed on mouse hepatocytes fixed by the Boehm-Sprenger procedure optimal for preserving the chromatin pattern. The question was whether fluorochromes specific to DNA make it possible to accurately quantitate the total nuclear DNA content when the chromatin pattern is preserved. Only HO and MA were found to provide satisfactory quantitation of nuclear DNA content, as assumed by both a small CV and a 4c to 2c ratio equal to 2. PI, EB, 7-AMD, and OM provided higher CV values, although the 4c to 2 c ratio was still equal to 2. QM, AO, CMA3, and DAPI provided non-reproducible and non-stoichiometric nuclear DNA content measurements under the fixation conditions used. The intranuclear and the internuclear SD of the fluorescence intensities describing the fluorescence pattern of the 2c hepatocytes proved to vary according to both the basepair specificity and the binding mode of the fluorochromes. The results reported here argue in favor of an external binding of 7-AMD to DNA and an increased quantum yield of QM when bound to AT-rich DNA. For PI, EB, 7-AMD, and OM, the measured DNA content increased with the fluorescence distribution heterogeneity. This correlation was not observed with other fluorochromes and is suggested to result from decreased fluorochrome accessibility to DNA when the chromatin is condensed. This study demonstrates that under conditions that preserve chromatin organization, only HO for AT-rich DNA and MA for GC-rich DNA can be used, alone or in combination, to measure nuclear DNA content. With other fluorochromes, either the measured DNA content or the chromatin pattern is assessed in suboptimal conditions when fluorescent image cytometry is used.     

Highly chlorinated Escherichia coli cannot be stained by propidium iodide

Several studies have shown that the staining by fluorochromes (DAPI, SYBR Green II, and TOTO-1) of bacteria is altered by chlorination. To evaluate the effect of chlorine (bleach solution) on propidium iodide (PI) staining, we studied Escherichia coli in suspension and biomolecules in solution (DNA, RNA, BSA, palmitic acid, and dextran) first subjected to chlorine and then neutralized by sodium thiosulphate. The suspensions and solutions were subsequently stained with PI. The fluorescence intensity of the PI-stained DNA and RNA in solution dramatically decreased with an increase in the chlorine concentration applied. These results explain the fact that for chlorine concentrations higher than 3 micromol/L Cl2, the E. coli cells were too damaged to be properly stained by PI. In the case of highly chlorinated bacteria, it was impossible to distinguish healthy cells (with a PI-impermeable membrane and undamaged nucleic acids), which were nonfluorescent after PI staining, from cells severely injured by chlorine (with a PI-permeable membrane and damaged nucleic acids) that were also nonfluorescent, as PI penetrated but did not stain chlorinated nucleic acids. Our results suggest that it would be prudent to be cautious in interpreting the results of PI staining, as PI false-negative cells (cells with compromised membranes but not stained by PI because of nucleic acid damage caused by chlorine) are obtained as a result of nucleic acid damage, leading to an underestimation of truly dead bacteria.     

Consequences of stoichiometric error on nuclear DNA content evaluation in Coffea liberica var. dewevrei using DAPI and propidium iodide

The genome size of coffee trees (Coffea sp.) was assessed using flow cytometry. Nuclear DNA was stained with two dyes [4',6-diamino-2-phenylindole dihydrochloride hydrate (DAPI) and propidium iodide (PI)]. Fluorescence in coffee tree nuclei (C-PI or C-DAPI) was compared with that of the standard, petunia (P-PI or P-DAPI). If there is no stoichiometric error, then the ratio between fluorescence of the target nuclei and that of the standard nuclei (R-PI or R-DAPI) is expected to be proportional to the genome size. Between-tree differences in target : standard fluorescence ratios were noted in Coffea liberica var. dewevrei using propidium iodide and DAPI. For both dyes, between-tree differences were due to a lack of proportionality when comparing locations of the coffee peak and the petunia peak. Intraspecific genome size variations clearly cannot explain variations in the target : standard fluorescence ratio. The origin of the lack of proportionality between target and standard fluorescences differed for the two dyes. With propidium iodide, there was a regression line convergence point, and no between-tree differences were noted in this respect, whereas there was no such convergence with DAPI. An accurate estimate of genome size can thus be obtained with PI. Implications with respect to accessibility and binding mode are discussed.     

Compare Analysis for the Nanotoxicity Effects of Different Amounts of Endocytic Iron Oxide Nanoparticles at Single Cell Level

Developing methods that evaluate the cellular uptake of magnetic nanoparticles (MNPs) and nanotoxicity effects at single-cellular level are needed. In this study, magnetophoresis combining fluorescence based cytotoxicity assay was proposed to assess the viability and the single-cellular MNPs uptake simultaneously. Malignant cells (SKHep-1, HepG2, HeLa) were incubated with 10 nm anionic iron oxide nanoparticles. Prussian blue stain was performed to visualize the distribution of magnetic nanoparticles. MTT and fluorescence based assay analyzed the cytotoxicity effects of the bulk cell population and single cell, respectively. DAPI/PI stained was applied to evaluate death mechanism. The number of intracellular MNPs was found to be strongly correlated with the cell death. Significant differences between cellular MNP uptake in living and dead cells were observed. The method could be useful for future study of the nanotoxicity induced by MNPs.     

Counterstaining human chromosomes for flow karyology

Isolated human metaphase chromosomes stained with the fluorochromes 4'-6-diamidino-2-phenylindole (DAPI) and chromomycin A3(CA3), and counterstained with nonfluorescent netropsin (NTR), have been analyzed by dual-laser flow cytometry. Counterstaining with NTR reduces DAPI fluorescence except at regions on chromosomes 1,9,15,16, and Y, corresponding to C-band heterochromatin. Bivariate flow karyology of human chromosomes treated with this triple-stain combination resolves chromosomes 1,9, and Y distinctly from the remaining chromosomes and resolves variations between chromosome homologues not detected by staining with propidium iodide (PI) or with the double stain combination Hoechst 33258(HO) and CA3.     

Schistosoma mansoni: protein composition and synthesis during early development; evidence for early synthesis of heat shock proteins

There are alterations in the proteins synthesized during different stages of development of Schistosoma mansoni. The protein profiles at different stages were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When stained by Coomassie blue, no significant differences were seen in protein profiles derived from cultured schistosomula from Days 0 to 6 and from adults. Newly synthesized proteins were detected by [35S]methionine incorporation. There were only a few differences in the protein profiles of schistosomula from Days 1 to 6 and from adults. Profiles derived from Day 0 schistosomula showed striking differences. Only a few proteins appear to be synthesized on Day 0 under these conditions. Schistosomula on Day 0 synthesized several minor proteins as well as a major protein of approximately 69,000 Da. This protein was immunoprecipitated by rabbit antiserum against bovine uncoating adenosine triphosphatase which recognizes the constitutive and induced 70,000 Da heat shock proteins in a wide variety of eukaryotic cells. More significant differences were observed when the newly synthesized proteins were analyzed by two dimensional gel electrophoresis. The profiles of newly synthesized proteins showed a specific repertoire of expression during the early stages of development in the parasite. A shift in temperature and medium during transformation from aquarium water to isotonic medium may initiate the synthesis of heat shock protein in these parasites.     

Two-step cell-death kinetics in vitro during cis-platinum, hydroxyurea and mitomycin incubation

A human leukaemic cell line (REH) growing in suspension was incubated with cis-platinum, hydroxyurea and mitomycin C at various concentrations causing complete cell-cycle arrest. At different times the cell suspensions were harvested, diluted 1:1 with a buffer solution, stained without further treatment with a mixture of acridine orange (AO) and ethidium bromide (EB) and analysed with a biparametrical flow cytometer. Fluorescent plastic beads were introduced into the suspensions to provide an internal numerical reference for the control of cell loss. The fluorescence distributions showed three groups of cells: vital cells (V) which were only stained with AO; dead cells in which EB stained cytoplasmic components but not the nuclear DNA (D1), and dead cells which allowed EB to stain both cytoplasm and nuclear DNA (D2). The kinetics of cells entering D1 depended on drug concentration and showed equal characteristics for cis-platinum and mitomycin, but were different for hydroxyurea. The subsequent entry into D2 occurred about 15 hr later and showed no pronounced dependence on drug concentration. Parallel trypan-blue (TB) exclusion tests revealed that TB only stained D2 cells and therefore is not useful for investigating cell-death kinetics during exposure to cell-killing agents.     

A monoclonal antibody raised against adult Schistosoma mansoni which recognizes a surface antigen on schistosomula

A monoclonal antibody has been raised by immunizing a mouse with an isolated tegumental preparation of adult Schistosoma mansoni. The hybridoma designated NIMP/M.47, secreted an IgG2a antibody which was positive by indirect immunofluorescence with live schistosomula of S. mansoni, but not with live schistosomula of S. bovis, or with other living life cycle stages of S. mansoni. In complement-dependent, or cell-mediated in vitro cytotoxicity assays, the monoclonal antibody mediated levels of schistosomular killing as high as those obtained with sera from infected mice. No significant protection, however, was obtained in passive transfer experiments. NIMP/M47 was specific for a 20,000 dalton polypeptide in the schistosomular surface, which was also recognized by serum from infected mice.     

Two-Step Cell-Death Kinetics In Vitro During Cis-Platinum, Hydroxyurea and Mitomycin Incubation

A human leukaemic cell line (REH) growing in suspension was incubated with cis-platinum, hydroxyurea and mitomycin C at various concentrations causing complete cell-cycle arrest. At different times the cell suspensions were harvested, diluted 1:1 with a buffer solution, stained without further treatment with a mixture of acridine orange (AO) and ethidium bromide (EB) and analysed with a biparametrical flow cytometer. Fluorescent plastic beads were introduced into the suspensions to provide an internal numerical reference for the control of cell loss. The fluorescence distributions showed three groups of cells: vital cells (V) which were only stained with AO; dead cells in which EB stained cytoplasmic components but not the nuclear DNA (D1), and dead cells which allowed EB to stain both cytoplasm and nuclear DNA (D2). the kinetics of cells entering D1 depended on drug concentration and showed equal characteristics for cis-platinum and mitomycin, but were different for hydroxyurea. the subsequent entry into D2 occurred about 15 hr later and showed no pronounced dependence on drug concentration. Parallel trypan-blue (TB) exclusion tests revealed that TB only stained D2 cells and therefore is not useful for investigating cell-death kinetics during exposure to cell-killing agents.     

Schistosomula of Schistosoma mansoni clear concanavalin A from their surface by sloughing

The lectin concanavalin A (Con A) was used as a model probe to study the behavior of molecules bound to the surface of recently transformed schistosomula of Schistosoma mansoni. Con A binding was saturable (150- 180 pg/organism) and specifically competed by alpha-methyl mannoside. Both FITC-Con A and 125-I-Con A were lost from the surface of schistosomula with a halftime of 8-10 h in culture in defined medium. A comparable decrease in the binding of Con A to schistosomula cultured and then labeled with the lectin indicated that the labeling procedure itself was not inducing the observed change. Internalization of Con A was not seen by either fluorescence microscopy or electron microscope radioautography. In addition, 70-80% of the radioactivity lost from the parasite was recoverable by TCA precipitation from the culture medium as intact Con A (27,000 mol wt on SDS PAGE). Thus, the mechanism of clearance of bound Con A from the surface of cultured schistosomula is apparently by sloughing of Con A molecules intact into the culture media and not by endocytosis and degradation. Con A binding sites, visualized with hemocyanin by scanning electron microscopy, appeared homogeneously distributed over the surface of schistosomula when organisms were labeled at 4 degree C or after fixation with glutaraldehyde. However, Con A and hemocyanin formed aggregates on the surface of schistosomula when labeling was performed at 37 degrees C, which suggests that lectin binding sites have lateral mobility within the plane of the membrane. These aggregates are likely independent of metabolism by the parasite because aggregation also occurs on the surface of organisms killed with azide.     

Schistosoma mansoni: a comparative study of artificially transformed schistosomula and schistosomula recovered after cercarial penetration of isolated skin.

A comparison was made of the ultrastructure, development and antigenic nature of the surfaces and of the viability of three types of Schistosoma mansoni: schistosomula formed after cercariae had penetrated isolated skin (SS) schistosomula produced after mechanical separation of cercarial tails from bodies (MS), and schistosomula transformed from cercariae after incubation in fresh rat serum (RS). Within 2h of transformation, the surface membranes of all three types of schistosomula had changed from trilaminate to heptalaminate structures and SS and MS had lost their cercarial glycocalyx. Initially a dense amorphous material was demonstrated on the surfaces of RS, which was thought to be the result of an interaction between a factor in rat serum and the glycocalyx; this material was greatly reduced within 2 h of transformation. The pre-acetabular glands of SS were emptied while those of MS and RS retained their contents. Immunofluorescent studies showed that all schistosomula bound serum from mice immune to S. mansoni, but the binding was stronger with MS and RS. The mixed agglutination reaction demonstrated the presence of human A and B blood group-like antigenic determinants on approximately 30% of 3h old SS; these determinants were not detected on MS or RS. In vitro, the development of MS and RS was similar to SS; the first schistosomula reached the "gut-closed" stage by day 10; 50-70% of SS reached this stage by day 12, in contrast to only 25-50% of MS and RS. Between 28 and 45% of all schistosomula developed to maturity when injected intravenously into mice. It was concluded that the two types of artificially prepared schistosomula fulfil the main criteria of transformation from cercaria to schistosomulum. Further, it is suggested that MS are the most appropriate source of material for immunochemical and physiological studies.