bluuebunny/crossref_metadata_2025 huggingface dataset:
Fluorescent antibody localization of the vitamin D-dependent calcium-binding protein in the oviduct of the laying hen.
Authors: Lippiello L , Wasserman R H
Published: April 2011

Fluorescein isothiocyanate-conjugated sheep antirabbit gamma-globulin was used in the indirect fluorescent antibody technique to localize the vitamin D-dependent calcium-binding protein (CaBP) in the shell gland of the laying hen. Intense specific fluorescence, indicative of the presence of CaBP, was found only in the tubular gland cells and not in the surface epithelium. CaBP was primarily associated with the luminal surface and other cell boundaries, and in lesser concentrations within the cells except for occasional intracellular regions of high concentration. The magnum, isthmus and vagina were essentially negative. Since the concentrations of intestinal CaBP is highly and directly correlated with the efficiency of calcium absorption, the present results suggest that the tubular gland cells might have a primary role in the transport of calcium associated with egg shell formation.
Rapid chemical dehydration of samples for electron microscopic examinations.
Authors: Muller L L , Jacks T J
Published: April 2011

Acidified 2,2-dimethoxypropane was used to chemically dehydrate biologic tissues for examination in the electron microscope. The ultrastructural integrities of single-celled algae, plant tissues (cotyledon, root, leaf) and animal tissues (liver, pancreas, muscle, cartilage) were maintained. Our technique was simpler and quicker than physically exchanging water for organic solvents (e.g., acetone, ethanol) as generally performed in microscopy.
Histochemical demonstration of mitochondrial dehydrogenases in developing normal and nervous mutant mouse Purkinje cells.
Authors: Landis S C
Published: April 2011

The mouse mutation nervous (nr), expressed as a recessive, causes a morphologic change in cerebellar Purkinje cell perikaryonal mitochondria during the 2nd postnatal week which is followed by the selective degeneration of most Purkinje cells. In the present study, mitochondrial dehydrogenase activities (succinic acid dehydrogenase, lactic acid dehydrogenase, malic acid dehydrogenase, reduced diphosphopyridine nucleotide diaphorase and succinic semialdehyde dehyrogenase) are found by light microscopy to be similar in the perikarya of normal and nervous Purkinje cells until after mitochondrial rounding has begun to occur, but then staining increases markedly in nervous. Although only a small fraction of mitochondrial functions were examined, those enzymes assayed were present and competent. In a sense, the only mitochondrial abnormalities demonstrated thus far in nervous Purkinje cells are their unusual shape and probable loss of motility.
Polysaccharides as labels for antibodies in electron microscopy.
Authors: Kent S P , Wilson D V
Published: April 2011

Two polysaccharides, dextran 250 and dextran 70, were covalently linked to antibody molecules, antihuman immunoglobulin G and antihuman type O red blood cells. In electron microscope preparations exposed to lead citrate, polysaccharides, because they chelate lead, were quite dense. Polysaccharides served as a tag for the antibody molecules. Also, bacterial dextran 1355 was used to demonstrate antibody molecules on the surface of ascites tumor cells which are known to be producing a specific antibody to bacterial dextran 1355. The varying sized polysaccharide molecules that are readily available commercially, the high electron density of the polysaccharides after lead staining and a mild procedure for covalently linking polysaccharide to antibody make polysaccharides attractive as particulate labels for antibody in electronmicroscopy.
Colloidal alpha-stannic acid and negative iron colloid as differential electron stains for surface proteins.
Authors: Blanquet P R , Puvion E
Published: April 2011

Colloidal alpha-stannic acid and a negative iron colloid obtained from ferric hydroxide and potassium ferrocyanide, both negative sols being stable within a wide pH range, were refined as surface protein electron markers. Because of the relatively small size of its particles, colloidal alpha-stannic acid was used for staining all surface proteins. According to the pH at which the negative iron colloid was applied, it revealed either all surface proteins, or because of its large colloidal particles, stained basic proteins. This differential staining capability of the iron colloidal has been demonstrated previously on various control preparations (Puvion E, Blanquet PR: J Microsc 12:171, 1971). Controls on the affinity of the two colloids to surface amino groups were carried out on rat liver, mouse fibroblasts, HeLa and KB cells, Ehrlich and Zajdela ascites cells subjected to prior enzymatic and chemical treatments (incubation with neuraminidase or phospholipase C, esterification, acetylation or lipid extraction). At any pH below 9, the two sols stained proteins in the outer hydrophilic leaflet of esterified cells with relative selectivity, but the alpha-stannic acid showed them more accurately. The iron sol did reveal at high pH protein components of high isoionic point on the surfaces of rat hepatocytes and ascites cells which had only been treated with neuraminidase.
The ultrastructural cytochemistry of lactic dehydrogenase, succinic dehydrogenase, dihydro-nicotinamide adenine dinucleotide diaphorase and cytochrome oxidase activities in hair cell mitochondria of the guinea pig cochlea.
Authors: Spector G J
Published: April 2011

The use of cinnamyl nitroblue tetrazolium chloride (DS-NBT) in dehydrogenase experiments (lactic dehydrogenase, succinic dehydrogenase, nicotinamide adenine dinucleotide diaphorase) and 3,3'-diaminobenzidine tetrahydrochloride (DAB) in cytochrome oxidase experiments indicated that mitochondrial oxidoreduction reactions from nicotinamide adenine dinucleotide to cytochrome oxidase are located on the inner mitochondrial membrane in the outer compartment and the intracristate spaces. These reactions behave according to the chemiosmotic hypothesis. The cochlear hair cell mitochondria are cytochemically indistinguishable from free liver mitochondria. The heterogeneous mitochondrial staining pattern is related to the osmolarity of the incubation media, solubility of the enzymes and pH of the medium, but not to the fixation method.
Histochemical localization of cytochrome oxidase in gastric mucosa.
Authors: Hershey S J , Simon T W , Baste C
Published: April 2011

A cytochemical technique for localizing cytochrome oxidase activity, based upon the oxidative polymerization of 3,3-diaminobenzidine (DAB) to an osmiophilic reaction product, has been employed to test the possibility of extramitochondrial cytochrome oxidase in gastric mucosa. Deposition of reaction product was found to be exclusively localized within mitochondris; in particular, no reaction product was observed at the apical plasma membrane. Measurements of the effect of DAB on acid secretion revealed a biphasic action consisting of an initial stimulation followed by inhibition. The stimulation of secretion of DAB is interpreted to indicate that DAB is oxidized via a pathway which is linked to the secretory process. The combined cytochemical and physiological measurements provide evidence that the metabolic energy supply for acid secreation is derived from mitochondrial reactions. The results are discussed in relation to current models for the coupling between acid secreation and oxidative metabolism.
Differential feulgen-deoxyribonucleic acid hydrolysis patterns of Herpes simplex virus type 1 and type 2 infected cells.
Authors: Trusal L R , Anthony A , Docherty J J
Published: April 2011

Infection of human embryonic lung cells with herpes simplex virus type 1 (HSV-1) and herpes simplex type 1 (HSV-2) resulted in: (a) qualitative (nuclear cytopathologic) alterations and quantitative (nuclear area) differences in infected compared to control nuclei; (b) increased Feulgen-deoxyribonucleic acid (F-DNA) amounts in infected cells, probably due to viral DNA; (c) higher F-DNA levels in HSV-2 infected cells; and (d) increased rates of F-DNA hydrolysis in viral-infected as compared to uninfected nuclei.
Ultrastructural localization of endogenous mammary gland peroxidase during lactogenesis in the rat results after tannic acid-formaldehyde-glutaraldehyde fixation.
Authors: Anderson W A , Trantalis J , Kang Y H
Published: April 2011

Endogenous mannary gland peroxidase in acinar cells of prelactating and lactating rats is revealed in tannic acid-formaldehyde-glutaraldehyde-fixed tissue by means of the standard diaminobenzidine procedure. Diaminobenzidine cytochemical reaction product is present in perinuclear cisternae, in the granular endoplasmic reticulum and in Golgi apparatus of functionally differentiated secretory cells. The mammary gland peroxidase is thought to represent lactoperoxidase. Peroxidase staining is diminished or absent in acinar cells of hypophysectomized and ovariectomized rats, in normal rats during early pregnancy and in nonpregnant mature females. Endogenous peroxidase or a heme protein with peroxidatic activity may be considered an ultracytochemical marker enzyme for acinar cells actively engaged in lactogenesis.
Effects of fixation and postfixation treatments on volume of injured cells;.
Authors: Penttila A , McDowell E M , Trump B F
Published: April 2011

The effects of fixation with glutaraldehyde (GA), formaldehyde (FA), glutaraldehyde-formaldehyde (GA-FA), flutaraldehyde-osmium tetroxide (GA-Os04) and osmium tetroxide (OsO4) on cel volume were studied in control, p-chloromercuribenzene sulfonic acid (PCMBS)-treated and hypotonically-treated Ehrlich ascites tumor cells. Among the variables investigated were concentration of the tixative agent, osmolity of the buffer, total osmolaity of the fixation solution, osmolaltity of the postfixation buffer and the time of fixation and postfixation treatment; in addition, the effects of adding calcium and high molecular weight compounds to the fixative solution were studied. When the effects of standard fixatives on control, PCMBS- and hypotonically-treated cells were compared, marked differences were apparent in the behavior of control and injured cells. Control cells retained near prefixation volume in 3% GA and 3% GA-1% OsO4, swelled in 4% FA or 1% OsO4 and phosphate buffer (tkrp), whereas tpcmbs (310 mosM KRP)- and hypotonically-treated cells (103 mos M KRP) shrank in all aldehyde fixatives but swelled in 1% OsO4. Reducing the buffer osmolality had similar effects on control and injured cells although, there were variations in degree...
Buoyant density separation of cells. I. The buoyant distribution of guinea pig bone marrow cells.
Authors: Leif R C , Smith S B , Dunlap L A , Leif S B
Published: April 2011

Guinea pig bone marrow cells were separated by buoyant density utilizing linear gradients of bovine serum albumin (BSA). It has finally become possible to characterize the cells present in the density fractions in terms of classical morphology. The development of the Cell Type computer program which calculates the percentages of the individual types of cells present in the fractions and their buoyant density distributions and plots the data has greatly facilitated and improved the accuracy of these studies. Approximately 40 cell types were observed in guinea pig bone marrow. Cells with definitive morphologies such as erythrocytes, the neutrophilic series, the binucleate blast megakaryocyte precursor and cells in mitosis band as virtually single peaks. Cells which are parts of continua or can easily be wrongly classified are found in multiple peaks. The small lymphocytes which are known to be polydisperse are found as five peaks. Because of the very strong benzidine staining by the glutaraldehyde-fixed hemoglobin, some of the erythroblasts were wrongly staged, resulting in a multimodal distribution. The presence of macrocytes further complicated these distributions. The rule that the younger cells are always less dense than the mature cells was adhered to in those cases where the cells could be definitively characterized, such as the neutrophilic series and the blasts. These results indicate that morphology is a good first approximation of reality.
Acid-beta-glycerophosphatase reaction products in the central nervous system mitochondria following x-ray irradiation.
Authors: Roizin L , Orlovskaja D , Liu J C , Carsten A L
Published: April 2011

A survey of the literature to date on the enzyme histochemistry of intracellular organelles has not yielded any reference to the presence of acid phosphatase reaction products in the mammalian mitochondria of the central nervous system. A combination of Gomori's acid phosphatase mehtod, however, with standard electron microscopy has disclosed the presence of enzyme reaction products in the mitochondria of the central nervous system of rats from 2 hr to 22 weeks after x-ray irradiation, as well as in a cerebral biopsy performed on a patient affected by Huntington's chorea. No enzyme reaction products, on the other hand, were observed in serial sections that had been incubated in substrates either containing sodium fluoride or lacking in beta-glycerophosphate. The abnormal mitochondrial enzyme reaction (chemical lesion) is considered to be the consequenco of the pathologic process affecting the ultrastructural-chemical organization of the organelle.
Demonstration of lysosomal and extralysosomal sites for acid phosphatase in mouse kidney tubule cells with p-nitrophenylphosphate lead-salt technique.
Authors: Miyayama H , Solomon R , Sasaki M , Lin C W , Fishman W H
Published: April 2011

Dual localization of acid phosphatase in lysosomal and extralysosomal sites of the tubule epithelial cells of normal mouse kidney was observed at the light and electron microscope level using a modified Gomori lead-salt method with p-nitrophenylphosphate (pNPP) as substrate. Based on previous biochemical and cytochemical findings, we developed optimal conditions for the enzyme activity in extralysosomal sites. The conditions used for the light microscopic level consisted of 1.5 mM PNPP, 2.0 MM Pb(NO3)2 and 0.05 M acetate buffer (pH 5.8). Those for the electron microscopic study required 3.0 mM PNPP, 3.6 MM Pb(NO3)2 and 0.1 M acetate buffer (pH 5.8). This modified lead-salt technique was highly specific and provided a suitable method for the demonstration of nonlysosomal as well as lysosomal sites of acid phosphatase activity in the tubule epithelial cells of normal mouse kidney. As expected, the enzyme activity appeared in the lysosomes, but the prominent reaction in the brush border, the rough endoplasmic reticulum and basal infolding plasma membranes was not anticipated. We were able to demonstrate in situ organelle precursors of microsomal acid phosphatase such as endoplasmic reticulum, plasma membrane and basal infolding membranes showing the same substrate preference, which had been observed previously in biochemical studies in our laboratory. Since the possible participation of alkaline phosphatases, K+-pNPPase or Na+-K+-adenosine triphosphatase was ruled out by use of appropriate inhibitors, the enzyme-reactive sites can be interpreted as reflecting nonspecific acid phosphatase.
Acid treatment of Drosophila deoxyribonucleic acid.
Authors: Sederoff R , Lowenstein L , Mayer A , Stone J , Birnboim H C
Published: April 2011

In cytologic preparations of chromosomes, acid-treated deoxyribonucleic acid (DNA) is found largely in the native state. However, acid treatments widely used for chromosome preparations produce significant amounts of depurination in DNA. DNA is similarly sensitive to depurination in intact cells or as purified DNA. If treated with alkali, these apurinic gaps can be converted to single strand breaks. Acid treatment has widely different effects on specific fractions of DNA. In Drosophila melanogaster 3% of the DNA is composed of very long tracts of pyrimidines (polypyrimidines) which are resistant to acid hydrolysis. The implications of these results for molecular cytogenetics are discussed.
Distribution of concanavalin A binding sites on the surface of dissociated rat submandibular gland acinar cells.
Authors: Amakawa T , Barka T
Published: April 2011

The submandibular glands of 4-week-old rats were dissociated by a procedure involving digestions with collagenase and hyaluronidase, chelation of divalent cations and mechanical force. A suspension of single cells was obtained in low yield by centrifugation in a Ficoll-containing medium. Immediately after dissociation and after a culture period of 16-18 hr the dissociated cells were tested for agglutinability by concanavalin A (Con A). Using ferritin (tfer)-conjugated Con A the lectin binding by the isolated acinar cells was also studied. The dissociated cells were agglutinated by low concentrations of Con A and bound Fer-Con A molecules on their entire surface without any indication of polarization of the cell membrane. There was a considerable cell to cell variation in the amount of Fer-Con A binding which was, in general, sparse and patchy. The contact surfaces between agglutinated cells revealed a dense binding of Fer-Con A molecules irrespective of the types of cells participating in the agglutination reaction. Cells cultured for 16-18 hr were no longer agglutinated by Con A. As compared to the freshly dissociated cells the cultured acinar cells revealed a more uniform and denser binding of Fer-Con A molecules. Furthermore, there were more lectin molecules bound to the cell surface corresponding to the basal part of the cell, where the nucleus and most of the rough surface endoplasmic reticulum were located, than to the apical cell surface. It is suggested that the higher density of lectin-binding sites on the cell surface in the vicinity of the cisternae of the rough endoplasmic reticulum indicates insertion sites of newly synthesized membrane glycoproteins.
The significance of inhibitor-resistant alkaline phosphatase in the cytochemical demonstration of transport adenosine triphosphatase.
Authors: Firth J A , Marland B Y
Published: April 2011

The hydrolysis of disodium p-nitrophenyl phosphate at pH 9.0 by slices of formaldehydee-fixed rat renal cortex was investigated by colorimetric estimation of the nitrophenol liberated. It was found that three types of activity could be identified on the basis of their responses to inhibitors and cations: (a) alkaline phosphatase sensitive to inhibition by L-tetramisole; (b) potassium-dependent phosphatase, probably identifiable with the phosphatase component of sodium-potassium-dependent transport adenosine triphosphatase (?Na-K-ATPase); and (c) alkaline phosphatase insensitive to L-tetramisole. It was found that in the presence of strontium ions, as used in Na-K-ATPase cytochemistry, the activities of the second and third types of enzyme were approximately equal. The implications of these findings for the cytochemical demonstration of Na-K-ATPase are discussed.
Ultrastructural localization of gamma-chain and immunoglobulin G in human lymphocytes using enzyme-labeled Fab fragment.
Authors: Lin C T , Chang J P , Chen J P
Published: April 2011

By the use of rabbit antibodies against the heavy chain of human immunoglobulin G (IgG), the gamma-chain and IgG molecules were successfully localized at the ultrastructural level in human peripheral lymphocytes. The rabbit Fab fragment was coupled to horseradish peroxidase by means of glutaraldehyde and the resulting conjugate could penetrate the intact plasma membrane. Discernible reaction product was observed in cisternae of the nuclear envelope, rough endoplasmic reticulum and Golgi apparatus as well as on the surface of the lymphocytes. In normal human individuals under no specific antigenic stimulation, only a few peripheral lymphocytes showed a rare positive intractoplasmic reaction. Reaction product may represent either the whole IgG molecule, the half molecule consisting of one heavy and one light chain or nascent gamma-chain.
Enzymes of the cholinergic system in islets of Langerhans.
Authors: Godfrey D A , Matschinsky F M
Published: April 2011

Quantitative radiometric assays were employed to measure activities of choline acetyltransferase and acetylcholinesterase in freeze-dried pieces of islets of Langerhans and exocrine tissue from rat pancreas. The activities of both enzymes were about an order of magnitude higher in islets than in exocrine tissue. This difference in activity was found in rats made diabetic with streptozotocin as well as in the controls. Although the enzyme activities in islets from diabetic rats averaged about 30-40% higher than those in islets from control rats, the differences were statistically only marginally significant. Since the islets of diabetic rats are probably much smaller than those of control rats, it is suggested that cholinergic elements associated with pancreatic islets are lost following induction of streptozotocin diabetes.
Stoichiometry of the aldehyde fuchsin staining reaction for proteins.
Authors: Mahon D C , Nair K K
Published: April 2011

Model systems of agar films containing known concentrations of bovine serum albumin and alpha-chymotrypsinogen were stained with aldehyde fuchsin after oxidation with acidified permanganate solution. These films were scanned in a scanning microphotometer to determine the mean extinction and the total extinction of predetermined areas. Results indicate that the dye binds quantitatively to the proteins. Blocking the acidic side groups of the proteins inhibited the binding of the dye. The degree of inhibition was directly related to the number of sulfhydryl or carboxyl groups that were blocked. Similar blocking reactions performed on the type "A" neurosecretory cells of the pars intercerebralis of the insect Oncopeltus fasciatus gave similar results. Analysis of the dye protein complexes gave a dye to acidic group ratio of 1:1.
An automated microscope for cytologic research a preliminary evaluation.
Authors: Brenner J F , Dew B S , Horton J B , King T , Neurath P W , Selles W D
Published: April 2011

A research-oriented system for automated microscopy is described from an operational point of view. The system consists of a microscope, a TV camera, an automatic cell finder and a servo-driven computer controlled stage. The system is interfaced to a NOVA 840 computer having 112,000 words of 16-bit core memory and extensive peripherals. It is capable of performing a wide variety of image processing tasks and is being used to study various aspects of automated microscopy, with applications in, but not limited to, cytology. Results of preliminary performance evaluations are given.
Methods of quantitative autoradiography using incident light microphotometry.
Authors: Dörmer P , Thiel E
Published: April 2011

Several different approaches of automated grain counting of microautoradiographic grain densities have been reported in the literature. Application of grain counters to cell biology is limited, however, primarily due to shortage of methods allowing the interpretation of grain counts on a molecular basis. Two suitable methods of quantitative autoradiography at the cellular level are reviewed, developed for the isotopes 14C and 125 I. They permit evaluation of absolute radioactivity in autoradiographs and, thus, determination of synthesis processes such as deoxyribonucleic acid synthesis, and of antigen densities on cell surfaces. In this approach towards quantitative autoradiography, grain densities are compared photometrically over labeled cells and over a standard source on the same autoradiograph. Allowance has to be made for the specific geometric factors of the isotope used. This can be advantageously done with an integrating type of measurement using incident light bright-field. With this type of recording, there is an exponential dependence of the photometric values on the radioactive dose. As an example of application, results are presented of the deoxyribonucleic acid synthesis rate of human myelocytes in aplastic anemia and of the immunoglobulin G density on lymphocyte membranes in the normal state and in chronic lymphocytic leukemia.
Image processing for automated erythrocyte classification.
Authors: Bacus J W , Belanger M G , Aggarwal R K , Trobaugh F E
Published: April 2011

Digital image processing and pattern recognition techniques were applied to determine the feasibility of a natural n-space subgrouping of normal and abnormal peripheral blood erythrocytes into well separated categories. The data consisted of 325 digitized red cells from 11 different cell classes. The analysis resulted in five features: (a) size, (b) roundness, (c) spicularity, (d) eccentricity and (e) central gray level distribution. These features separated the data into six distinct condensed subgroups of red cells. Each subgroup consisted of morphologically similar cells: (a) macrocytes, (b) normocytes, (c) schistocytes, acanthocytes and burr cells, (d) microcytes and spherocytes, (e) elliptocytes, sickle cells and pencil forms and (f) target cells. The concept of a quantitative "red cell differential" was introduced, utilizing these subgroup definitions to establish subpopulations of red cells, with quantifiable indices for the diagnosis of anemia, at the specimen level.
Basic principles of electrical sizing of cells and particles and their realization in the new instrument "Metricell".
Authors: Kachel V
Published: April 2011

To understand the basic events during the passage of particles through the Coulter orifice, three experiments were performed. (1) The denpendence of the volume results on the particle path has been shown by ink-colored particle beams. (2) The deformation and alignment of cells during their passage through the orifice have been photographed by a nano-second photographing technique. (3) The absolute volume evaluation of particles has been studied with model particles in enlarged model orifices of different lengths. A new compact sizing instrument, "Metricell," equipped with a particle-independent electrical calibrating system, is described.
Estimation of cell size from pulse shape in flow cytofluorometry.
Authors: Sharpless T K , Melamed M R
Published: April 2011

The fluorescence pulse widths (pulse duration) generated by fluorochromed cells in a flow-through cytofluorometer provide useful information regarding cell (or nuclear) size and possibly other morphologic features. Simple fixed thresholds just above background noise can be used to identify these pulses, but measurements are then strongly affected by random noise and will vary as a result of both pulse amplitude and pulse shape. In this paper, we propose two alternative, amplitude-independent estimates of pulse width. The first is based on a threshold at some fraction of pulse height, or on a pair of thresholds scaled to some fixed central fraction of the total integrated intensity. The second is based on the ratio of pulse area to peak height. The quantitative properties of these width estimators is studied with simulated fluorescence pulses and with experimental specimens of fluorchromed polystyrene spheres, pollen and spores of known different diameters. The results indicated that absolute particle diameters can be measured within a precision of approximately 1 mu using instruments for flow cytofluorometry.
Flow microfluorometric and light-scatter measurement of nuclear and cytoplasmic size in mammalian cells.
Authors: Steinkamp J A , Hansen K M , Crissman H A
Published: April 2011

A technique for rapid measurement of nuclear and cytoplasmic size relationships in mammalian cell populations has been developed. Based on fluorescence staining of either the nucleus alone or in combination with the cytoplasm using two-color fluorescence methods, this technique permits the simultaneous determination of nuclear and cytoplasmic diameters from fluorescence and light-scatter measurements. Cells stained in liquid suspension pass through a flow chamber at a constant velocity, intersecting a laser beam which excites cell fluorescence and causes light scatter. Depending upon which analysis procedure is used, optical sensors measure nuclear fluorescence and light scatter (whole cell size) or two-color nuclear and cytoplasmic fluorescence from individual cells crossing the laser beam. The time durations of signals generated by the nucleus and cytoplasm are converted electronically into signals proportional to the respective diameters and are displayed as frequency distribution hitograms. Illustrative examples of measurements on uniform microspheres, cultured mammalian cells and human exfoliated gynecologic cells are presented.
Studies of cellular differentiation by automated cell separation. Two model systems: Friend virus-transformed cells and Hydra attenuata.
Authors: Arndt-Jovin D J , Ostertag W , Eisen H , Klimek F , Jovin T M
Published: April 2011

The automated high-speed analysis and separation of cells on the basis of spectroscopic parameters has been applied to studies of cellular differentiation in two systems. The temporal changes following induction of differentiation by dimethylsulfoxide in the Friend virus-transformed erythroid cells were quantitated by multiparameter analysis leading to the separation of discrete subpopulations. Thus, following induction, cell size decreased as measured by light scattering, the number of H-2 histocompatibility antigen sites decreased as measured by indirect fluorescent antibody binding, the number of lectin-binding sites per cell increased as measured by fluorescein-labeled concanavalin-A and the microviscosity of the hydrocarbon region of the plasma membrane increased as determined by the fluorescence emission anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene. Cells were separated on the basis of several of these parameters and analyzed for their hemogloglobin content by benzidine staining. Examination of cells separated according to the anisotropy parameter showed that high anisotropy values were correlated with (a) small cell size, (b) positive staining with benzidine and (c) pronounced reactivity with fluorescent antibody to the erythrocyte protein spectrin. Disaggregated cells from Hydra attenuata were selectively stained with the dyes rhodanile blue, 7-(p-methoxybenzylamino)-4-nitrobenz-2-oxa-1,3-diazole and fluorescamine. Distribution analyses and preliminary separations indicated the feasibility of obtaining homogeneous classes of cell types in a viable state. The experiments with emission anisotropy represent the first analyses and separations of single cells on the basis of fluorescence polarization. Many other uses of this technique are anticipated.
Quantitation of lymphocyte response to antigen by flow cytofluorometry.
Authors: Braunstein J D , Good R A , Hansen J A , Sharpless T K , Melamed M R
Published: April 2011

The response of human peripheral blood lymphocytes to antigenic stimulation has been studied in vitro using flow cytofluorometry and an acridine orange (AO) staining technique for cellular deoxyribonucleic acid and ribonucleic acid (RNA). Antigen-stimulated "pyroninophillic" immunoblasts, identified by an increase in cellular content of RNA (red fluorescence with AO), were quantitated in triplicate cultures incubated up to 7 days with and without bacterial antigen. These results were similar to 14C-thymidine incorporation into identical cultures incubated in parallel. Cytofluorometric analysis showed a peak in percentage of immunoblasts after 6 days in culture, while maximum thymidine incorporation was seen on day 7. Cells from patients with depressed immune response secondary to cancer showed lower than normal antigen response by cytofluorometry. Kinetic studies revealed both a lower percentage of immunoblasts when compared to normal and a lower average per cell RNA content of the stimulated cells. AO cytofluorometry is suggested as a convenient method of simultaneously assessing lymphocyte proliferative and nonproliferative response to antigen.
Cytofluorometric studies on conformation of nucleic acids in situ. I. Restriction of acridine orange binding by chromatin proteins.
Authors: Traganos F , Darzynkiewicz Z , Sharpless T , Melamed M R
Published: April 2011

Binding of the fluorochrome acridine orange (AO) to nucleic acids in situ is studied by automated cytofluorometry in two differentiating cell systems: Friend virus-transformed murine erythroleukemia induced to differentiate by dimethyl sulfoxide, and phytohemagglutinin-stimulated human lymphocytes. The specificity of the stain for deoxyribonucleic acid is discussed on the basis of data obtained by cell treatment with nucleases. Evidence is presented that in the case of Friend leukemia cells, but not phytohemagglutinin-stimulated lymphocytes, a significant change in the number of AO-intercalating sites in DNA occurrs during differentiation. These results suggest that changes in nuclear chromatin occurring during cell differentiation may be correlated, in some but not all systems, with changes in accessibility of DNA in situ to intercalating dyes. The role of divalent cations, especially Mg2+, in the conformation of nuclear chromatin and in modulation of the accessibility of nucleic acids to AO is discussed. The method provides a tool for the study of nucleic acid-protein interaction in situ, and in some cell systems it may be applicable as a marker for recognition of cell transformation, differentiation or neoplasia.
Cell electrophoresis research directed toward clinical cytodiagnosis.
Authors: Boltz R C , Todd P , Gaines R A , Milito R P , Docherty J J , Thompson C J , Notter M F , Richardson L S , Mortel R
Published: April 2011

The application of cell electrophoresis to cytodiagnosis requires that a scientifically established basis exists for identifying abnormal cells electrophoretically, that research to detect such differences in the cytodiagnostic setting is possible and that a rapid and simple method of cell electrophoresis is adaptable to the clinical setting. Data are presented indicating modifications of electrophoretic mobility due to herpes simplex virus type 1 infection and Rous sarcoma virus transformation of culture cells. A simple apparatus for electrophoretically separating cells on a density gradient and collecting them for subsequent analysis is described, and results of experiments with this apparatus are consistent with those obtained by microscopic electrophoresis. Laser-doppler spectroscopic electrophoresis is suggested as a rapid method adaptable to clinical application.
Quantitative fluorescence spectrophotometry of acridine orange-stained unfixed cells. Potential for automated detection of human uterine cancer.
Authors: Golden J F , West S S , Echols C K , Shingleton H M
Published: April 2011

After staining with acridine orange (AO), the nuclei of unfixed cells from the human female genital tract exhibited the same fluorescence behavior previously observed for human and murine leukocytes and mouse ascites tumor cells. With staining conditions chosen to assure saturation of the green-fluorescing AO-nucleic acid complex in normal cells, corrected fluorescence emission spectra were recorded from the entire nucleus of 341 cells taken from 32 normal and 28 abnormal patients. Intensity of the recorded spectra was expressed in phosphor particle units, a fixed arbitrary unit of fluorescence intensity, to display intensity differences among the spectra from the various cell types. In all abnormal samples, one or more cells were found with 530-nm nuclear fluorescence intensity considerably greater than the maximum intensity recorded from normal cells. Determination of the adequacy of 530-nm nuclear fluorescence intensity as a criterion for cancer detection requires additional investigation. Additional criteria, if needed, may be supplied by the metachromasy of AO-stained unfixed cells.
Studies on human monocytes with a multiparameter cell sorter.
Authors: Kwan D , Epstein M B , Norman A
Published: April 2011

Suspensions of human lymphocytes and monocytes separated by the Ficoll-hypaque method from the peripheral blood show a Coulter volume distribution, measured with a multiparameter cell sorter, characterized by a minor peak at 500 mu3, containing 5-15% of the cells, and a major peak at 200 mu3. Using fluorescent latex particles we have found that the monocytes, the cells that ingest the latex particles, all lie in the 500 mu3 peak; conversely, all of the cells in the 500 mu3 peak are monocytes. When the cell suspensions are incubated, the monocytes increase both the average volume and in absolute numbers. The number of monocytes approximately doubles during 3 days of incubation, when it reaches its maximum value. At that time we have found that all of the monocytes lack receptors for sheep red blood cells and all possess receptors for human gamma-globulin. The increase in monocyte number appears, therefore, to arise from the enlargement of "monocyte presursors" that resemble lymphocytes in volume and resemble both the monocytes and the B lyphocytes with respect to surface sheep red blood cell and human gamma-globulin receptors.
Combined blood cell counting and classification with fluorochrome stains and flow instrumentation.
Authors: Shapiro H M , Schildkraut E R , Curbelo R , Laird C W , Turner B , Hirschfeld T
Published: April 2011

A multiparameter flow cytophotometer was used to count and classify fixed human blood cells fluorochromed with a mixture of ethidium bromide (EB), brilliant sulfaflavine and a blue fluorescent stilbene disulfonic acid derivative (LN). The system measures light scattered by the cells and absorption at 420 nm for all cells. In addition, nuclear EB fluorescence (540 leads to 610 nm) and cytoplasmic fluorescence from LN (366 leads to 470 nm), brilliant sulfaflavine (420 leads to 520 nm) and EB exicted by energy transfer from LN (366 leads to 610 nm) are measured for all nucleated cells. This information is sufficient to perform red and white blood cell counts and to classify leukocytes as lymphocytes, monocytes, basophils, eosinophils or neutrophils. Light scattering and/or nuclear and cytoplasmic fluorescence values may be further analyzed to obtain the ratio of immature to mature neutrophils. Counts produced by the system are in reasonable agreement with those obtained by electronic cells counting and examination of Wright's-stained blood smears; some discrepancies appear to be due to systematic errors in the manual counting method.
Rapid staining methods for analysis of deoxyribonucleic acid and protein in mammalian cells.
Authors: Crissman H A , Oka M S , Steinkamp J A
Published: April 2011

Quantitative fluorescent staining and analysis of cellular deoxyribonucleic acid (DNA) were accomplished using three groups of reagents having different mechanisms of action for DNA binding. These reagents included (a) the fluorescent antitumor antibiotics mithramycin, chromomycin A3 and olivomycin; (b) the Feulgen reagents acriflavine and flavophosphine N and (c) the intercalating dyes ethidium bromide and propidium iodide. Propidium iodide (PI) was used in combination with fluorescein isothiocyanate (FITC) to stain both cellular DNA and protein, respectively. Multiparameter analysis of PI/FITC-stained cells provided a direct correlation of DNA and protein for cells in all stages of the cell cycle. Nuclear-to-cytoplasmic ratio determinations were also performed on PI/FITC-stained cells by analysis of the time duration of the red (DNA) and green (protein) fluorescence signals from each cell. These staining and analysis techniques provide alternative methods for directly determining the quantitative relationship between cellular DNA and protein and will be extremely useful in investigations where fluctuations of these parameters are of importance for assessing experimental results.
A new type of two-color fluorescence staining for cytology specimens.
Authors: Cornelisse C J , Ploem J S
Published: April 2011

A new two-color fluorescence staining technique for cervical cytology specimens is described. To permit application of this staining in automated cytology, techniques for specimen collection and cell preparation giving a sufficient number of well-separated cells on slides were used. The staining consists of a combination of a modified Feulgen-acriflavine procedure for DNA and a primulin or stilbene isothiocyanate staining for protein. This results in a bright yellow nuclear fluorescence and a blue cytoplasmic fluorescence. The staining procedure can be completed in about 90 min and is therefore suitable for routine application. Sequential inspection of the yellow nuclear and blue cytoplasmic fluorescence can be done with the two-wavelength excitation method used in fluorescence microscopy. For the application of this method, special vertical illuminators are now available. These illuminators are provided with quickly interchangeable filter sets permitting consecutive visualization of, for example, only the nuclei in the first image and the whole cell in the second image. This procedure opens new possibilities for rapid image-analysis systems.
Flow microfluorometric quantitation of the blastogenic response of lymphocytes.
Authors: Cram L S , Gomez E R , Thoen C O , Forslund J C , Jett J H
Published: April 2011

A method for quantitating the specific stimulation of peripheral lymphocytes has been developed using the techniques of flow microfluorometry. Peripheral bovine lymphocytes were collected and specifically stained for deoxyribonucleic acid (DNA) content using a low-salt propidium iodide procedure. Flow microfluorometry was used to determine, on the basis of DNA content, the percentage of cells in a population that was stimulated. Extremely uniform staining of the lymphocytes (coefficient of variation of less than 2%) provides a high resolution between proliferating and nonproliferating cells. The method provides a rapid, highly repoducible technique for determing the fraction of lymphocytes stimulated in response to tuberculin antigens based on an increase in cellular DNA content. Specific and nonspecific stimulation by a defined antigen can be measured and resolved.
Variation in the autoradiographic technique: I. Emulsion-developer combinations assessed by photometric measurement of single silver grains.
Authors: Neely J E , Combs J W
Published: April 2011

Five commonly used developers (Acufine, Rodinal, Dektol, D19 and Microdol X) and two popular nuclear gel emulsions (Kodak NTB-2 and Ilford L4) were tested in combinations for adequacy of development characteristics and the distribution of silver particle (grain) size measured by means of a reflectance microphotometer. All developer-emulsion combinations had some range of development time during which maximum development was accomplished with no occurrence of background grains, with the exceptions of the combinations of NTB2-Acufine and NTB2-D19. Ilford L4 emulsion obtained the narrowest grain size distribution with Rodinal and Acufine, followed by Dektol, and D19. The narrowest size distribution for Kodak NTB2 emulsion was achieved with D19 followed by Acufine, Dektol and Rodinal. Microdol-X had undesirable effects on the integrity of the individual grains of both emulsions. The criteria are discussed for selecting the most advantageous emulsion-developer system for the particular mode of evaluation (visual, photometric, television camera) to be applied to the finished autoradiographic specimens.
A comparative ultrastructural localization of concanavalin A, wheat germ and Ricinus communis on glomeruli of normal rat kidney.
Authors: Bretton R , Bariety J
Published: April 2011

Three lectins, concanavalin A, peroxidase labeled wheat germ and peroxidase labeled Ricinus communis have been utilized to determine the localization of various saccharide determinants in the glomerulus of the normal rat kidney. In order to obtain a homogeneous penetration of the lectins throughout the whole section, various technical parameters were studied. Only with concanavalin A, a diffuse labeling of the endoplasmic reticulum was obtained. With the three lectins, the basement membrane, mesangial matrix and the cell coat of the three cell types in the glomerulus were labeled. Differences in the labeling of the capillary wall were also noted.
Localization of catecholamines in the eyes and other tissues of Aplysia.
Authors: Luborsky-Moore J L , Jacklet J W
Published: April 2011

A green fluorescence indicative of catecholamines (CA) was localized in the secondary cells (nonreceptor neurons), neuropile and optic nerve of the eye and other tissues in Aplysia by using the formaldehyde-induced fluorescence method for the demonstration of biogenic amines. The specificity of the induced fluorescence was confirmed by its absence in tissue not exposed to formaldehyde vapor, relatively rapid decay upon exposure to UV light and its chemical reduction by sodium borohydride. The fluorescence was greatly reduced in eyes treated with reserpine (depletes serotonin and catecholamines). Furhter confirmation that the green fluorescence in the eye was due to a CA and not to serotonin was obtained by showing that it was decreased or eliminated by alpha-methyl-para-tyrosine (an inhibitor of catecholamine synthesis), increased by incubation in dopamine and exhibited a peak emission (470 nm) characteristic of CA fluroescence. CA fluorescence was also observed in the neuropiles of the cerebral, pedal, pleural and parieto-visceral ganglia and in cells in the pedal ganglion, statocyst, mantle, anterior tentacles and siphon. The finding of CA in secondary neurons of the eye was unusual since CA-containing cells have not been observed in other gastropod eyes. The distribution of CA in Aplysia, in tissue other than the eye, is similar to that of other gastropod molluscs.
Cytochemical localization of glycoconjugate in mitochondria.
Authors: Parmley R T , Spicer S S , Poon K , Wright J
Published: April 2011

Complex carbohydrates and cations have been localized by cytochemical methods in mitochondria of mammalian leukocytes, hepatocytes and oyster gill epithelium. Glycoconjugate of acidic nature was visualized with the dialyzed iron method in or on the outer membrane and inner boundary membrane and in the outer intermembrane space but not on the membranes of cristae or in the intracristate space, or matrix. Sulfated glycoconjugate was demonstrated with the high iron diamine method in a similar distribution and in, on, or between, membranes of cristae as well. Intermittent aggregates of dialyzed iron and high iron diamine stained material were often found in the outer intermembrane space. The periphery of isolated rat liver mitochondria also stained with a Concanavalin A horseradish peroxidase technique, indicating the presence of macromolecules, presumably glycoproteins containing mannose or glucose. The distribution of antimonate reactive cation in mitochondria of leukocytes resembled that of the acidic glycoconjugate, indicating binding of cations to anionic groups of the latter. The complex carbohydrates and cations demonstrated cytochemically in mitochondria are considered in relation to previous biochemical studies.
The use of ferricyanide for the electron microscopic demonstration of dehydrogenases in human steroidogenic cells.
Authors: Benkoël L , Chamlian A , Barrat E , Laffargue P
Published: April 2011

The ultrastructural localization of 3 beta hydroxysteroid ferricyanide reductase, glucose-6-phosphate ferricyanide reductase and nicotinamide adenine dinucleotide and reduced form-ferricyanide reductase was investigated in some human steroidogenic tissues (corpus luteum of pregnancy, fetal adrenal gland and testis, adult testis and placenta) using ferricyanide as an electron acceptor. Copper ferrocyanide deposits were readily observed in the mitochondria, in the smooth endoplasmic reticulum profiles and in the cytoplasm. The sites of the various dehydrogenase activities could be visualized by using appropriate incubating media. The precise localization of various reactions in different electron transfer chains was determined by using different ferricyanide concentrations and intermediate electron-carriers such as menadione or exogenous nicotinamide adenine dinucleotide and reduced form-diaphorase. The use of respiratory chain inhibitors such as rotenone or antimycine A confirmed these data.