Triple labeling with two-color immunofluorescence using one light source: a useful approach for the analysis of cells positive for one label and negative for the other two

Many laboratories do not have access to a flow cytometer allowing three-color immunofluorescence analysis through the use of multiple light sources. In view of the usefulness of such analyses in the dissection of cell parameters, we describe an approach permitting the study of three labels by using one light source and the two-color immunofluorescence assay. It is useful for the enumeration of cell subpopulations positive for one label and negative for two or more others as well as for qualitative analysis concerning the expression of these labels. This approach is simple and rapid; it does not require additional material and technical steps other than that used in the two-color immunofluorescence assay. Briefly, it consists of the use of a label coupled to a dye (PE or FITC or instance) and two different labels coupled to the other dye. An argon ion laser, operating at 488 nm and 60 mW, excites both fluorescein and phycoerythrin conjugated antibodies. We provided a general example, using three hypothetical labels (X, Y, and Z), and four practical applications: CD3+CD4CD8- and CD8+CD16-CD3- peripheral blood lymphocytes, CD2+CD16-CD3- and CD56+CD16-CD3- peripheral blood, and decidual infiltrating lymphocytes.     

Simultaneous visualization of two antigens in the same tissue section by combining immunoperoxidase with immunofluorescence techniques.

A simple method was developed whereby immunoperoxidase and immunofluorescence techniques were applied in consecutive steps to demonstrate the presence of two antigens in the same tissue section. This method was applied in three model, two antigens were shown: a) each (gastrin and pepsinogen II) inside one of two different cell types (gastrin (G) and antral peptic cells), b) each (kappa or gamma light chains) inside different cells of the same type (plasma cells); also, both (kamma and gamma light chains) inside the same cell (Reed-Sternberg cell), and c) both (pepsinogen I and II) inside the same cell (chief cell of oxyntic glands). The results could be viewed and photographed either simultaneously, when the antigens were in different cells, or sequentially, when the antigens were in the same cells.     

Indirect immunofluorescence of microtubules in Dictyostelium discoideum. A study with polyclonal and monoclonal antibodies to tubulins

Amebae of D. discoideum on coverslips were fixed in situ with glutaraldehyde and permeabilized with Triton X-100. Of six antibodies tested, only a monoclonal antibody to yeast tubulin consistently gave bright fluorescence. Counterstaining with DAPI facilitated the identification of interphase and mitotic stages. Most microtubules (MTs) in interphase amebae emanated from a nucleus-associated centre that had a non-fluorescent core. Amebae in early stages of mitosis lacked cytoplasmic MTs almost entirely. The nascent spindle in prophase appeared as a brightly fluorescent dot, whereas the prometaphase spindle was a short rod. Spindles in metaphase and anaphase nuclei were more elongate, some consisting of several fluorescent lines. Astral MTs were prominent on spindles in anaphase and telophase. Asters are obviously converted to the interphase complex of MTs in post-mitotic cells, while the shaft-like remnant of the central spindle disappears. The cyclical changes in the MT system related to cell division resemble those observed in higher eukaryotes and probably reflect changes in the locomotory behavior of the amebae rather than changes in cell shape.     

Replication of human immunodeficiency virus in two T-cell lines and their application as antigens for immunofluorescence

Two T-cell lines, TALL-1 and CCRF-CEM, were infected with human immunodeficiency virus (HIV), strain LAV, to explore the time course of the appearance of various virus specific antigens, and to establish an antibody assay system by indirect immunofluorescence (IF). These cells were infected with LAV at two different input multiplicity of infection (MOI). Antigens were tested by Western blot analysis (WB) and IF. Antigens for WB were extracted from the infected cells at various times after infection, but pooled sera of American HIV carriers could not recognize gp41 or gp160. Antigen expression was highest in CCRF-CEM, but, as the antigen for IF, TALL-1 infected at the MOI of 8.0 was the most suitable 7 days after infection, because it includes a fairly large number of uninfected cells, which served as the internal control.     

Bioconversion of filter mud using vermicomposting employing two exotic and one local earthworm species

Three different earthworm species Eisenia fetida, Eudrilus eugeniae and Perionyx excavatus in individual (Monocultures) and combinations (Polycultures) were utilized to compare the suitability of worm species for vermicomposting of filter mud as well as the quality of the end product. The filter mud blended with saw dust can be directly converted into good quality fertilizer (vermicompost). Eight different reactors including three monocultures and four polycultures of E. fetida, E. eugeniae and P. excavatus and one control were used for the experiment. Vermicomposting resulted in significant reduction in C/N ratio, pH, total organic matter (TOC) but increase in electrical conductivity (EC), total nitrogen (TN), total phosphorus (TP) and macronutrients (K, Ca and Na). Oxygen uptake rate (OUR) dropped up to 1.64–1.95 mg/g (volatile solids) VS/day for monoculture reactors and 1.45–1.78 mg/g VS/day for polycultures reactors, respectively, after 45 days of vermicomposting. Cocoon production and the earthworm biomass increased as vermicomposting progressed. On an overall the mono as well as polyculture reactors produced high quality stable compost free from pathogens and no specific differentiation could be inferred between the reactors.     

Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function

Although a great deal is known biochemically about peroxiredoxins (Prxs), little is known about their real physiological function. We show here that two cytosolic yeast Prxs, cPrxI and II, which display diversity in structure and apparent molecular weights (MW), can act alternatively as peroxidases and molecular chaperones. The peroxidase function predominates in the lower MW forms, whereas the chaperone function predominates in the higher MW complexes. Oxidative stress and heat shock exposure of yeasts causes the protein structures of cPrxI and II to shift from low MW species to high MW complexes. This triggers a peroxidase-to-chaperone functional switch. These in vivo changes are primarily guided by the active peroxidase site residue, Cys(47), which serves as an efficient "H(2)O(2)-sensor" in the cells. The chaperone function of these proteins enhances yeast resistance to heat shock.     

Differences in the susceptibility to light stress in two lichens forming a phycosymbiodeme,one partner possessing and one lacking the xanthophyll cycle

Summary The effect of high light levels on the two partners of a Pseudocyphellaria phycosymbiodeme (Pseudocyphellaria rufovirescens, with a green phycobiont, and P. murrayi with a blue-green phycobiont), which naturally occurs in deep shade, was examined and found to differ between the partners. Green algae can rapidly accumulate zeaxanthin, which we suggest is involved in photoprotection, through the xanthophyll cycle. Blue-green algae lack this cycle, and P. murrayi did not contain or form any zeaxanthin under our experimental conditions. Upon illumination, the thallus lobes with green algae exhibited strong nonphotochemical fluorescence quenching indicative of the radiationless dissipation of excess excitation energy, whereas thallus lobes with blue-green algae did not possess this capacity. The reduction state of photosystem II was higher by approximately 30% at each PFD beyond the light-limiting range in the blue-green algal partner compared with the green algal partner. Furthermore, a 2-h exposure to high light levels resulted in large reductions in the efficiency of photosynthetic energy conversion which were rapidly reversible in the lichen with green algae, but were long-lasting in the lichen with blue-green algae. Changes in fluorescence characteristics indicated that the cause of the depression in photosynthetic energy conversion was a reversible increase in radiationless dissipation in the green algal partner and photoinhibitory damage in the blue-green algal partner. These findings represent further evidence that zeaxanthin is involved in the photoprotective dissipation of excessive excitation energy in photosynthetic membranes. The difference in the capacity for rapid zeaxanthin formation between the two partners of the Pseudocyphellaria phycosymbiodeme may be important in the habitat selection of the two species when living separate from one another.Abbreviations F _O yield of instantaneous fluorescence - F _M maximum yield of fluorescence induced by pulses of saturating light - F _V yield of variable fluorescence (F _M -F_O) induced by pulses of saturating light - PFD photon flux density (400–700 nm) - PS II photosystem II - q _NP coefficient for nonphotochemical fluorescence quenching - q _P (or 1-q _P ) coefficient for photochemical fluorescence quenching     

Three mouse monoclonal antibodies to human differentiation antigens: reactivity with two mucin-like antigens and with connective tissue fibers

Three human differentiation antigens (MU78, MT334, and MQ49) have been defined by mouse monoclonal antibodies developed from mice immunized with ovarian carcinoma cell lines. Their distribution was determined on 148 cultured cell lines of various histologic types and on frozen sections of 16 normal tissues. MU78 was found in fibrillar structures in soft connective tissue with a distribution resembling that of elastin fibers; however, elastin fibers in elastic cartilage and in the aorta were nonreactive. MU78 was detected in cultured carcinoma cells of various histologic types, where it had a nonfibrillar, cytoplasmic distribution, but was not detected in normal epithelial cells in frozen sections. Cultured fibroblasts, astrocytomas, melanomas, and lymphomas did not contain MU78. In cell lines, MU78 appears to be a protein of 2000-5000 daltons. The other two antigens, MT334 and MQ49, are both mucin-like molecules, and the determinants are probably carbohydrate in nature. Of the normal tissues examined, MT334 was detected only in goblet cells of the colon, though it was present in a variety of carcinomas in culture. It was detected as both a cytoplasmic and secreted component. MQ49 was detected in various secretory epithelial cells, in Hassall's corpuscles in the thymus, and in cultured carcinomas of various histologic types. It was found on the cell surface as well as in the cytoplasm and is present on a glycolipid as well as on a sulfated mucin. These results, and results of other recent studies, demonstrate the importance of mucin-like molecules as antigens in epithelial cells and secretions.     

Specificity of antigens from pathogenic Aspergillus species. I. Studies with ELISA and immunofluorescence

Studies were made by enzyme linked immunosorbent assay (ELISA) and indirect fluorescent antibody (IFA) tests on the reactivities and specificities of 13 antigens prepared from four species of Aspergillus against antisera from immunized rabbits and 64 sera from patients with aspergillosis, other systemic mycoses and nocardiosis. Although reactions in both serological tests were invariably strongest with homologous antigen: antibody systems, antisera from rabbits immunized with A. fumigatus, Blastomyces dermatitidis, Candida albicans and Paracoccidioides brasiliensis reacted in the ELISA test with all of the Aspergillus antigens. In contrast, cross-reactivity was virtually non-existent with antiserum to Histoplasma capsulatum. Of five antigens prepared from A fumigatus tested by ELISA against human sera from patients with aspergillosis and other nocardial and systemic fungal infections, sensitivities varied from 81 to 100% for sera from 32 patients with aspergillosis, and specificities from 20 to 97% for sera from 30 patients with nocardiosis and other systemic mycoses. Purified A. fumigatus C antigen reacted weakly with sera from eight of these 30 patients, but the reactions were readily distinguishable from those obtained with sera from patients with aspergillosis. At optimal serum dilutions, cross-reactivities of A. fumigatus in the IFA studies were non-existent in the sera from 28 patients with candidosis, coccidioidomycosis, cryptococcosis, histoplasmosis, paracoccidioidomycosis and nocardiosis. Sensitivities of IFA were 94% for patients with aspergilloma and 83% for patients with allergic bronchopulmonary aspergillosis.     

Interaction of chaotropically modified immunoglobulins with protein and glicolipid antigens

The features of interaction of native and chaotropically modified immunoglobulins with proteins (ovalbumin) and glicolipids (lipopolysaccharides, LPS) enterobacteria Escherichia coli K235, Salmonella minnesota and Salmonella enteritidis have been investigated. It has been established, that after processing of native antibodies with 3.5 M KSCN their ability to contact to the specified antigenes repeatedly grows. Besides the intensity of interaction of modified immunoglobulins with the mentioned above antigenes was various, that is determined by the presence of structural distinctions between antigen determinants of proteins and glycolipid antigens, and also between O-polysaccharide chains of LPS in different species of enterobacteria.     

Microspectrofluorimeter with an interference light filter of variable wavelength]

A highly sensitive portable microspectrofluorimeter with continuous interference filter for 400-700 nm spectrum, for investigation of living cells, is described.