Using dyes and filters in a fluorescent imaging system.

The FluorImager fluorescence imaging system uses monochromatic 488-nm laser light to excite fluorochromes. It contains a built-in 515-nm long-pass filter that rejects excitation laser light, but allows emission light with wavelengths longer than 515 nm to pass through. A fluorochrome appropriate for use in the system is excitable by 488-nm light and emits at least some of its fluorescence at wavelengths longer than 515 nm. Two types of optical filters, long-pass and band-pass, are used in the system. Long-pass filters reject shorter wavelengths and transmit longer wavelengths. The number in the filter name denotes the cutoff wavelength (midpoint of the transition between rejected and transmitted light) for the filter. Band-pass filters transmit a band of wavelengths and reject both shorter and longer wavelengths. The numbers in the filter name denote the center wavelength of the passed band and the width of the band at half maximum transmission. Generally, when scanning for a single fluorochrome, only the built-in 515-nm long-pass filter is needed. An interchangeable filter can be added to decrease the contribution from a broad-spectrum background signal and to attenuate strong fluorochrome signals.

Interaction of wild-type and catalytically inactive mutant forms of tissue-type plasminogen activator with human umbilical vein endothelial cell monolayers.

Several groups have demonstrated that radioiodinated tissue-type plasminogen activator (t-PA) binds to saturable sites on human umbilical vein endothelial cells (HUVECs) in culture (Hajjar, K. A., Hamel, N. M., Harpel, P. C., and Nachman, R. L. (1987) J. Clin. Invest. 80, 1712-1719; Beebe, D. P. (1987) Thromb. Res. 46, 241-254; Barnathan, E. S., Kuo, A., van der Keyl, H., McCrae, K. R., Larsen, G. L., and Cines, D. B. (1988) J. Biol. Chem. 263, 7792-7799). Here we report that most of the specific binding of 125I-t-PA to our HUVEC cultures is accounted for by binding to (i) plasminogen activator inhibitor type 1 (PAI-1), a t-PA inhibitor produced in abundance by HUVECs; and (ii) specific binding sites present on the plastic culture surface. The contribution of the sites on plastic can be eliminated by taking several precautions. Then, most or all of the specifically bound 125I-t-PA is present in a sodium dodecyl sulfate-stable 110-kDa 125I-t-PA.PAI-1 complex. Interestingly, a radioiodinated mutant form of t-PA, S478A, which is catalytically inactive and therefore unable to form the covalent complex with PAI-1, still binds to HUVECs. In fact, this ligand binds to HUVECs in 10-30-fold greater amounts than does wild-type 125I-t-PA (resulting in greater than 1 x 10(7) S478A 125I-t-PA molecules bound/cell at 12 nM ligand concentration). In contrast, diisopropyl fluorophosphate-treated t-PA binds to HUVECs in much smaller amounts than does wild-type t-PA. Several findings suggest that PAI-1 is a major binding site for S478A t-PA. The vast amount of binding observed with S478A t-PA, compared with wild-type t-PA, may be accounted for by an observed large scale release of wild-type 125I-t-PA.PAI-1 complexes from the solid phase (cells or extracellular matrix) into the culture medium. Immunoprecipitation experiments demonstrate that, in contrast to wild-type t-PA, S478A t-PA does not extract [35S]methionine-PAI antigen from metabolically labeled extracellular matrix. It is proposed that t-PA releases PAI-1 from the solid phase when it forms the irreversible covalent complex with the inhibitor, a process that does not occur with the catalytically inactive mutant form of t-PA.

Capacitation and acrosome reactions in human spermatozoa monitored by a chlortetracycline fluorescence assay.

Human spermatozoa were incubated in culture medium containing human serum albumin (HSA) to promote capacitation, which was monitored by a rapid chlortetracycline (CTC) fluorescence assay. Four CTC fluorescence patterns were readily distinguished, one of which appeared to be correlated with capacitated sperm. When capacitated sperm were treated with either ionophore A23187 or acid-solubilized mouse zonae pellucidae to induce the acrosome reaction, the CTC assay identified acrosome-reacted sperm by lack of fluorescence on the head. Fresh sperm would not undergo the induced acrosome reaction. The percentages of acrosome-reacted sperm identified by the CTC assay in induced and control populations were the same as those identified by the presently used indirect immunofluorescence and triple stain assays.

Soluble 80-kd fragment of cell-CAM 120/80 disrupts cell-cell adhesion.

Calcium-dependent cell adhesion molecules (CAMs) mediate intercellular adhesion in epithelial cells and in preimplantation mammalian embryos. One of these molecules, cell-CAM 120/80, is found on cells as a 120-kd membrane glycoprotein and as a soluble 80-kd species in conditioned culture medium [Damsky et al: Cell 34:455, 1983]. We have purified to homogeneity the soluble 80-kd fragment of cell-CAM 120/80 by using monoclonal antibody affinity chromatography. We have shown that the purified molecule can disrupt cell-cell adhesion in cultured epithelial cells, thus indicating that it is directly involved in the adhesive process. In addition, we have further characterized both the 120-kd cell-associated molecule and its 80-kd fragment, including N-terminal sequence analysis.

Identification and redistribution of lamins during nuclear differentiation in mouse spermatogenesis.

Chromatin may be attached to the nuclear envelope through interaction of the nuclear membrane lamins A, B, and C. Such a hypothesis requires that these proteins are present in all cells with chromatin attachment to the nuclear envelope. We have investigated the distribution of the lamins during spermatogenesis in mouse, which exhibits extremes in nuclear envelope structural changes. By immunohistochemical techniques using human auto-antibodies and monoclonal antibodies against these molecules, we found that the lamins persist through all stages of spermatogenesis, though in highly variable amounts. They are also present during meiotic prophase (pachytene) when chromosomes are only locally attached to the nuclear envelope, analogous to the early prophase of somatic cells. Restructuring of the early spermatid nuclear envelope is accompanied by the appearance of a new lamin at the acrosomal fossa. In the epididymal spermatozoon the distribution of different lamins varies markedly over the nucleus suggesting special structural functions. The presence of lamins throughout spermatogenesis supports the concept that they are a general feature of the nuclear envelope structure, even where a lamina is not recognizable ultrastructurally.

Biochemical characterization of the adhesion-related differentiation antigen XT-1.

The XT-1-molecule, an adhesion-related differentiation antigen of male mouse germ cells, is a 34 000 Mr glycoprotein with major charge isomer at pI 5.1 and is an integral component of the cell membrane. On large late pachytene spermatocyte, the molecule is present at a concentration of 2.5 X 10(3) molecules micron-2, which approximates HLA/ABC concentration on lymphocytes. By comparing the reactivity of four anti-XT-1 monoclonal antibodies, three of which elicit germ cell-germ cell adhesion, we have defined two distinct surface regions of the XT-1-molecule. The relationship of the XT-1-molecule with other known adhesion-related molecules and testicular antigens is discussed.

Induction of cell-cell adhesion by monovalent antibodies in a germ cell culture.

Four monoclonal antibodies, XT-I, MT-23, MT-24 and MT-29, that bind the XT-1-differentiation-antigen of male germ cells have been used to investigate the biological role of the XT-1-molecule of germ cells in short-term primary culture. Cultures from 10 days postpartum mice demonstrate increasing numbers of antigen-positive germ cells and increased antigen expression per cell with succeeding days of culture. Treatment of the antigen-positive cultures with three of the monoclonal antibodies, XT-I, MT-23 and MT-24, increases germ cell-germ cell adhesion in a dose-dependent fashion. Treatment with the fourth monoclonal antibody, MT-29, does not induce cell adhesion. The monovalent, Fab fragment of XT-I-antibody also elicits tight cell adhesion, thus ruling out antibody cross linking of molecules or cells. Saturating or near saturating amounts of the positive antibodies are required to produce adhesion, a result consistent with perturbation of a function that is performed by the sum of action of many of the XT-1-molecules on the cell. The ability of germ cells to undergo antibody-elicited tight adhesion is dependent on germ cell age and/or XT-1-antigen concentration. We hypothesize that the XT-1-molecule is involved in regulation of cell adhesion, an event which must occur in normal development.

Acrosomal status evaluation in human ejaculated sperm with monoclonal antibodies.

An important question in mammalian gamete physiology concerns how capacitation and the occurrence of acrosome reactions in motile sperm relate to fertility. Evaluation of these relationships has been restricted by practical limitations because rapid, quantitative assays are unavailable. We have developed a rapid, reproducible assay for the evaluation of acrosomal status utilizing monoclonal antibodies specific to antigens localized in the acrosomal cap region of the sperm head. Mice were immunized with human ejaculated sperm preparations and the resultant hybridomas producing antisperm antibody were selected by solid-phase radioimmunoassay and indirect immunofluorescence (IIF). Two monoclonal antibodies (HS-19, HS-21) recognized target antigens restricted to the acrosomal cap by IIF, and 87 +/- 8.5% of the sperm in fresh ejaculates from 10 different sperm donors showed positive cap fluorescence with these reagents. Loss of HS-21 binding as measured by IIF was correlated with disappearance of the acrosomal cap as observed directly by transmission electron microscopy. Acrosomal disappearance, artificially induced in vitro using the calcium ionophore A23187, also resulted in a loss of HS-21 binding. The induction of acrosomal loss by ionophore was dependent upon extracellular calcium. The data presented suggest that specific monoclonal antibodies can be used for the rapid evaluation of acrosomal status in mammalian sperm.

Enzymatic dissection of the functions of the mouse egg's receptor for sperm.

During the course of sperm-egg interaction in mice, zona pellucida glycoprotein ZP3 (approximately equal to 80 kDa) serves as both receptor for sperm (J. D. Bleil and P. M. Wassarman, 1980c, Cell 20, 873-882) and inducer of the acrosome reaction (J. D. Bleil and P. M. Wassarman, 1983, Dev. Biol. 95, 317-324). In this investigation, small ZP3 glycopeptides (approximately equal to 1.5-6 kDa), obtained by extensive digestion of the purified glycoprotein with insoluble Pronase, were assayed for both sperm receptor and acrosome reaction-inducing activities. While ZP3 glycopeptides were virtually as effective as intact ZP3 in inhibiting binding of sperm to eggs in vitro ("receptor activity"), unlike intact ZP3, they failed to induce sperm to undergo the acrosome reaction. The latter was determined by indirect immunofluorescence using a monoclonal antibody directed against the acrosomal cap region of sperm. These results suggest that the sperm receptor activity of ZP3 is dependent only on its carbohydrate components, whereas acrosome reaction-inducing activity is dependent on the polypeptide chain of ZP3 as well.

Characterization of a cell-surface differentiation antigen of mouse spermatogenesis: timing and localization of expression by immunohistochemistry using a monoclonal antibody.

The XT-1 antigen, bound by monoclonal antibody XT-I, is a differentiation antigen of germ cells in the mouse testis. As seen in immunoperoxidase-stained tissue sections from several juvenile ages and adult, the antigen becomes detectable on early (leptotene/zygotene) spermatocytes and increases in staining during spermatocyte development. During spermatid development the distribution of the determinant shifts from its relatively uniform surface distribution on spermatocytes to a more restricted localization on the base of the head, tail and cytoplasmic lobe of the elongating spermatid. The antigen is not detectable on juvenile or adult Sertoli cells. Detection of the antigen is dependent on the presence of germ cells of appropriate developmental stage. It is, thus, a marker for spermatocytes and later germ cells, for a cell-surface molecule related to spermatogenesis and for redistribution and/or modification of the molecule during spermatid elongation.

Characterization of human sperm surface antigens with monoclonal antibodies.

Monoclonal antibodies (McAb) against human ejaculated sperm were developed from mice immunized with sperm membrane preparations. A solid-phase radioimmunoassay, with dried sperm as antigen, was employed in McAb screening. The tissue and species specificity of monoclonal antibodies HS 2, 4 and 6 were evaluated after absorption of antibody preparations with heterologous sperm, human serum or seminal plasma or cells from other human organs. The sensitivity of HS 2, 4 and 6 antigens to trypsin exposure was determined: HS 4 antigen was highly sensitive while HS 2 and 6 were not. The regional distribution of McAb 4 on intact sperm cells was determined by immunofluorescence staining. HS 4 may be a sperm-coating antigen based on its presence on sperm and in seminal plasma. This possibility led to an investigation of its role in sperm capacitation. HS 4 antibody binding was reduced when capacitated sperm were compared with noncapacitated cells. HS 4 antibody, when present during capacitation and insemination, was without effect on sperm motility or fusion with zona-free hamster eggs. Trypsin removal of as much as 60% of HS 4 antigen from the cell population also did not impact on sperm function. To identify the molecular correlate of HS 4 antigen, membrane components were extracted from washed sperm with Nonidet P-40, concentrated by acetone precipitation and analyzed electrophoretically in SDS-urea on 10% polyacrylamide slab gels. Immunoassays on protein blots with peroxidase-coupled second antibody identified a single reactive species in the molecular weight range of 130,000. Multiple reactive components were detected in blot transfers of seminal plasma.

Detection of neuroblastoma cells in human bone marrow using a combination of monoclonal antibodies.

Two different types of monoclonal antibodies, antineuroblastoma (PI153/3), and antilymphocyte (P3B1-C3) were used to identify and classify tumor cells in the bone marrow of patients with neuroblastoma and with other types of cancer. Cells expressing the antigens were detected with peroxidase-coupled anti-Ig. The cell-surface labeling is manifested as a dense black precipitate at the membrane visualized by light microscopy. The combination of the two antibodies gives specific staining patterns for each cell type. PI153/3+, P3B1-C3- is specifically associated with neuroblastoma cells. PI153/3+,P3B1-C3+ is expressed on blast cells from some types of acute lymphoblastic leukemia and a small subpopulation of normal lymphocytes. These monoclonal antibodies thus allow specific visual detection of single neuroblastoma cells in bone marrow samples. The results demonstrate how combinations of monoclonal antibodies can be effectively used to identify specific cell types by their expression of and lack of specific marker determinants. Application of this principle is particularly relevant for dissecting populations of related cells and/or molecules.

Recognition of differentiation antigens of spermatogenesis in the mouse by using antibodies from spleen cell-myeloma hybrids after syngeneic immunization.

Two differentiation antigens of spermatogenesis in the mouse are defined by monoclonal antibodies from hybridomas produced between the myeloma P3-X63Ag8 and spleen lymphocytes immunized with syngeneic testis cells. These antigens are on testis cells, and not liver, kidney, brain, spleen, or whole ovary. They are species but not strain specific and trypsin sensitive but collagenase insensitive. On the basis of their appearance during the onset of spermatogenesis they appear to be differentiation antigens expressed during different but overlapping time windows during spermatogenesis.

Antibody response of C3H in equilibrium (CKB X CWB)F1 tetraparental mice to poly-L(Tyr,Glu)-poly-D,L-Ala-poly-L-Lys immunization.

To test whether the antigen-specific stimulation of low responder-genotype B cells in tetraparental mice is due to a histoincompatibility reaction (allogeneic effect) against these B cells, tetraparental mice were constructed (a) between an Ir-1A low responder to the antigen poly-L(Tyr,Glu)-poly-D,L-Ala--poly-L-Lys. [(T,G)-A--L] and an Ir-1A F1 high responder and (b) between two histoincompatible Ir-lA low responders. In the first case the F1 high responder embryo shares the whole of the H-2 complex, including Ir, with the low responder embryo.

Genetic control of the antibody response to poly-L(Tyr,Glu)-poly-D,L-Ala--poly-L-Lys in C3H--CWB tetraparental mice.

In order to further delineate the mechanisms underlying genetic unresponsiveness, tetraparental mice were constructed from immune response-1A gene high responder and low responder parental genotypes, then were immunized with poly-L-(Tyr,Glu)-poly-D,L-Ala--poly-L-Lys ((T,G)-A--L). An analysis of the total serum allotype mixture and of the antigen-binding capacity of the separated allotypes demonstrated that in the milieu of a tetraparental mouse, both high and low responder B cells could be stimulated equally to produce identical high titered anti-(T,G)-A--L responses. Furthermore, these studies show that effective stimulation could occur across a histocompatibility disparity.