The use of dyes in reagents for radioimmunoassays.
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Coloring Agents↗
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A semi-automated system for the assessment of toxicity to cultured mammalian cells based on detection of changes in staining properties.
We have established a semi-automated microtiter-based system for the quantification of dye binding to cultured eukaryotic cells. This system has been applied to the quantitation of toxic activities that disrupt cell monolayers and their neutralization. We have used this background as a basis for developing a detection and characterization system for activities that do not cause such gross toxicity. A prototype system has been established based on three staining procedures which in broad terms assess cellular dehydrogenase activity, and protein, DNA, and RNA content. The activity of several agents affecting cyclic nucleotide metabolism, including cholera toxin, on the staining properties of exposed monolayers has been assessed. Several new categories of cellular response are readily discernible in this latter system indicating that biological activities may be identified on the basis of the pattern of such responses. Since microtiter based systems show considerable potential for automation, it is suggested that the further development of this approach could offer a realistic prospect for numerous forms of toxicity testing on an industrial scale.
Animals↗
Homogeneous, micelle quenching fluoroimmunoassay for detecting amphetamines in urine.
We developed a homogeneous fluoroimmunoassay for detecting amphetamines in urine. Only fluorescence intensity need be measured because the emission of non-protein-bound fluorescein-labeled amphetamine is preferentially quenched by detergent micelles. In a previous reported prototype assay system for measuring gentamicin in serum we used fluorescein and dodecyl sulfate (Anal Chem 1985; 57:1928-30). We have found that favorable hydrophobic and (or) ionic character of the analyte and unfavorable polar and (or) ionic character of the fluor are important determinants of the desired interactions. An anionic detergent and fluorescein, therefore, should be appropriate for apolar of cationic analytes, such as gentamicin and amphetamines. A greater [H+] at the anionic micelle surface is important for quenching emission from the fluor moiety. Millimolar concentrations of dodecyl sulfate rapidly denature immunoglobulin unless hapten is bound with sufficiently high affinity. Affinity was sufficiently high for the antibody used in the prototype gentamicin assay but not for the amphetamine antibody. Thus for the amphetamine assay, we used a non-denaturing detergent, dodecyl(oxyethylene)12 sulfate. The assay requires 30 microL of specimen in 2 mL of total assay volume. Amphetamine(d-,dl-, and meth-), at a concentration of 1 mg per liter of urine, is readily detected.
Amphetamine↗
[Photodynamic effect of quinacrine on bacteria].
The acridine dye quinacrine (QA) was tested with regard to the photodynamic action on bacteria (Proteus mirabilis, Escherichia coli). The absorption maximum of the yellow dye QA ist in contrary to the photodynamically active dyes methylene blue (MB) and thiopyronine (TP) situated in the short wave region of the visible spectrum. Using for illumination a common light source--they have in general a weak emission in the short wave region--relatively high concentrations of QA are necessary for photodynamic action, and the difference between photodynamic inactivation and toxic effect is small. Using that light source XBO 500 with nearly equal emission in the range from 400 to 700 nm, a distinct photodynamic action of QA results. Comparing the photodynamic action of QA with those of MB and TP, QA has a low photodynamic effect, and the kinetics of inactivation of bacteria with QA is completely different from those obtained with the dyes MB and TP.
Escherichia coli↗
Functional lymphocytes in the chicken pineal gland.
The primary antibody response of lymphoid tissue occupying the pineal gland of 6-wk-old chickens was studied subsequent to injection of the carotid artery with sheep red blood cells (SRBC) or bovine serum albumin (BSA). Injection of SRBC did not produce plaque-forming cells (PFC) among pineal lymphocytes whereas BSA stimulated synthesis of anti-BSA immunoglobulin in pineal lymphoid tissue. A cytotoxic assay using appropriate anti-lymphocyte sera indicated that single-cell suspensions of pineal lymphocytes were composed of 42% B lymphocytes and 51% T lymphocytes. Bursal and thymic lymphocytes labeled with tritiated thymidine migrated into pineal lymphoid tissue when injected into 4- and 5-wk-old naive chicks. These observations indicate that the bursa and thymus make equivalent contributions to the lymphoid mass in the chicken pineal gland. Challenge of pineal-established lymphocytes by antigen introduced via the blood vascular system suggests that soluble antigens--rather than particulate ones--stimulate antibody production in the pineal gland. Collectively, these studies indicate that the pineal gland should be considered as a functional component of the chicken's lymphomyeloid system.
Animals↗
Thiopyronine-sensitized photodynamic effect on RNA synthesis in Saccharomyces cells in vivo.
The effect of photodynamic treatment with thiopyronine and visible light on RNA metabolism in yeast cells was investigated at different times during logarithmic growth. The results show that RNA synthesis in the nucleus of the cells is not directly inhibited photodynamically. In the endoplasmic reticulum of photodynamically treated cells one finds mRNA in about the same relative amounts and quality as in untreated control cells, but the binding of polysomes on membranes in the cytoplasm as the first step of protein synthesis is inhibited for a long time after treatment as well as the synthesis of ribosomes.
Endoplasmic Reticulum↗
Immunosuppressive effect of concanavalin A. IV. The effect of Con A on the local graft versus host (GvH) reaction in the mouse popliteal lymph node.
In the applied parental F1 hybrid system, the GvH character of the regional lymph node reaction was checked against syngeneic and the absolute control systems. It was found that a cortical B-cell zone of the recipients did not participate in the acute phase of the GvH reaction. Cytologically, the GvH response consisted in a significant increase in large pyroninophilic cells, histologically - in a cellular proliferation and pyroninophilia, as well as in a marked angiogenesis beyond the cortical zone of the lymph node. The injection of Con A into the F1 hybrid mice, 24 hours before the administration of the parental cells, inhibited the earlier (presumably donor origin) cellular reaction and intensified the later (presumably host origin) response.
Animals↗
[Stimulation of post-traumatic regeneration of rat spleen under conditions of gravitational stress].
Post-traumatic regeneration of the rat spleen was studied after resection of half the organ, under gravitation overloading (11 units) using spleen tissue extract prepared by Filatov's method. Gravitation overloading caused a decrease in the size of the spleen nodules, smooths their contours, increases the red pulp infiltration by the lymphocytes, reduces the number of labeled cells and the intensity of the label in the reactive centers of the spleen nodules, decelerates the capsule formation in the resection area. Application of the stimulant normalized the structure of white pulp, increased the number of labeled cells, and accelerated the capsule formation. The tissue extract used in gravitation overloading brought the restoration process nearer to the usual course of the posttraumatic spleen regeneration (by the character and periods of tissue differentiation development).
Animals↗
Profile of cell cycle in hematopoietic malignancy by DNA/RNA quantitation using 7AAD/PY.
Using 7-amino-actinomycin-D/pyronin Y (7AAD/PY), we analyzed the surface phenotypes and cell cycle of 22 hematopoietic cell lines based on their cellular DNA/RNA content. Populations of G1a, G1b, S, and G2M, the DNA index (DI), and the RNA index of S phase (SRI) were calculated by means of DNA/RNA dot plots. Two new parameters were extracted from the cell-cycle profiles: the nucleic acid index of S phase (NI) and the coefficient of variations in the RNA at S phase (SVC). DNA/RNA dot plots of cell lines revealed four characteristic profiles of the cell cycle, defined with the calculated NI and SCV. These were type 0 (small NI, large SCV), type I (small NI, small SCV), type II (large NI, small SCV), and type III (large NI, large SCV). Type O included four stem cell lines: one t(1;19) leukemia, two Ph1+ acute lymphocytic leukemia (ALL), and one biphenotypic crisis of chronic granulocytic leukemia (CGL). Type I included five ALL cell lines: three T-ALL and two common B-ALL. Type II contained 10 myeloid cell lines: five AML and five myeloid crisis of CGL. Type III contained three relatively immature lymphoma cell lines: two Burkitt's lymphoma and one follicular center lymphoma. Calculated NI/SCV (%) were as follows: type 0, 2.27 +/- 0.19/16.7 +/- 3.7; type I, 2.20 +/- 0.30/11.1 +/- 0.7; type II, 3.64 +/- 0.52/11.8 +/- 1.0; and type III, 3.60 +/- 0.53/17.5 +/- 1.9. Cell-cycle analysis of blasts using 7AAD/PY combined with surface phenotyping may yield important information for classifying hematopoietic malignancy within 2 hours of patient admission.
Antigens, CD↗
Nuclear dyes and cytoplasmic staining.
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Apoptosis↗