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At least 19 recordsLinked to original sources

PubMed1992

Determination of glomerular size-selectivity in the normal rat with Ficoll.

Diffusion studies in vitro indicate that Ficoll behaves more like an ideal spherical molecule than does dextran, suggesting that Ficoll would be a better probe of glomerular pore size than the commonly used dextran. To examine the differences between these macromolecules in vivo, the fractional clearances of tritiated Ficoll and dextran were measured over a wide range of molecular sizes (Stokes-Einstein radius, rs, from 19 to 65 A) in normal euvolemic Munich-Wistar rats. Whole-kidney and single-nephron hemodynamic conditions were characterized through a combination of clearance and micropuncture measurements. The fractional clearance, or sieving coefficient (theta), for dextran significantly exceeded that of Ficoll at all molecular sizes examined, theta for dextran being approximately 10 times that for Ficoll for rs greater than 30 A. Thus, the results with Ficoll imply a more size-restrictive barrier than do the results with dextran. The values of theta for Ficoll approximated previously reported values for uncharged globular proteins. Although theta for Ficoll at rs = 35 A was much smaller than the corresponding value for dextran, it was still approximately 30 times greater than typical values of the filtrate-to-plasma concentration ratio reported for serum albumin (a polyanion) in the rat, in agreement with the concept that glomerular charge-selectivity normally plays an important role in the prevention of albuminuria. Three membrane-pore models were compared in their ability to represent the dextran and Ficoll sieving data. A lognormal pore-size distribution in parallel with a nonselective "shunt" pathway was found to be more effective than either an isoporous membrane with a shunt or a purely lognormal distribution. On the basis of these laboratory results and computations, Ficoll may be preferred over dextran in future studies of glomerular size-selectivity.

Animals↗

PubMed1990

Tracer diffusion in F-actin and Ficoll mixtures. Toward a model for cytoplasm.

We have previously reported that self-diffusion of inert tracer particles in the cytoplasm of living Swiss 3T3 cells is hindered in a size-dependent manner (Luby-Phelps, K., D.L. Taylor, and F. Lanni. 1986. J. Cell Biol. 102:2015-2022; Luby-Phelps, K., P.E. Castle, D.L. Taylor, and F. Lanni. 1987. Proc Natl. Acad. Sci. USA. 84:4910-4913). Lacking a theory that completely explains our data, we are attempting to understand the molecular architecture responsible for this phenomenon by studying tracer diffusion in simple, reconstituted model systems. This report contains our findings on tracer diffusion in concentrated solutions of Ficoll 70 or Ficoll 400, in solutions of entangled F-actin filaments, and in solutions of entangled F-actin containing a background of concentrated Ficoll particles or concentrated bovine serum albumin (BSA). A series of size-fractionated fluorescein-Ficolls were used as tracer particles. By fluorescence recovery after photobleaching (FRAP), we obtained the mean diffusion coefficients in a dilute, aqueous reference phase (Do), the mean diffusion coefficients in the model matrices (D), and the mean hydrodynamic radii (RH) for selected tracer fractions. For each model matrix, the results were compared with similar data obtained from living cells. As in concentrated solutions of globular proteins (Luby-Phelps et al., 1987), D/Do was not significantly size-dependent in concentrated solutions of Ficoll 400 or Ficoll 70. In contrast, D/Do decreased monotonically with increasing RH in solutions of F-actin ranging in concentration from 1 to 12 mg/ml. This size dependence was most pronounced at higher F-actin concentrations. However, the shape of the curve and the extrapolated value of D/Do in the limit, RH----O did not closely resemble the cellular data for tracers in the same size range (3 less than RH less than 30 nm). In mixtures of F-actin and Ficoll or F-actin and BSA, D/Do was well approximated by D/Do for the same concentration of F-actin alone multiplied by D/Do for the same concentrations of Ficoll or BSA alone. Based on these results, it is possible to model the submicroscopic architecture of cytoplasm in living cells as a densely entangled filament network (perhaps made up of F-actin and other filamentous structures) interpenetrated by a fluid phase crowded with globular macromolecules, which in cytoplasm would be primarily proteins.

Actins↗

PubMed1978

Synergistical effects of ficoll and phytohemagglutinin on human lymphocytes.

The influence of increasing concentration of the highly polymerized dectran Ficoll on cultured peripheral human blood lymphocytes stimulated or not stimulated by PHA was studied. The incorporation of 14C-thymidine into acid-insoluble material of unstimulated lymphocytes has not been influenced in the presence of increasing concentrations of Ficoll either pretreated with chelating resin or not pretreated. Ficoll not pretreated with chelating resin potentiates the PHA-induced stimulation by the factor 4.3 at 0.1 mg Ficoll/ml culture medium, and by the factor 3.2 at 1.0 mg/ml when PHA stimulated HPBL were used. Ficoll after pretreatment with chelating resin does not influence the DNA-synthesis at 0.1 mg Ficoll/ml medium, but causes a drop of the incorporation of 14C-thymidine by the factor 2.0 at 1.0 mg/ml. Using 10.0 mg per ml, both Ficoll preparation cause a decrease of the DNA-synthesis by the factor 2.5--3.0--probably a cytotoxic effect. The results obtained with emission spectrographic analysis and conductivity measurements show, that Ficoll after treatment with chelating resin contains about 10 times more ions (Mg, Ca, Na, Si) and has a 3.2-fold higher conductivity then Ficoll without pretreatment. It is possible that phenomena like electrochemical changes on the surface of the lymphocytes and osmotic alterations in the culture medium are responsible for these effects.

Cells, Cultured↗

PubMed1992

Priming of peripheral lymph node B cells with TNP-Ficoll: role of lymphokines in B cell differentiation.

We have previously shown that peripheral lymph node (PLN) B lymphocytes of adult DBA/2J mice failed to make an antibody response to type 2 antigen TNP-Ficoll, but exhibited a good antibody response to type 1 antigen TNP-Brucella abortus. In the present study we wanted to find out whether the unresponsiveness of PLN B cells to TNP-Ficoll is due to defects in the early activation and proliferation stage or in the final differentiation stage of B cells. Therefore, we have used a two-step protocol of in vivo immunization of mice with TNP-Ficoll and the subsequent in vitro challenge with TNP-Brucella abortus and studied the anti-TNP plaque-forming cell (PFC) responses. The results indicate a three- to sixfold increase of PFC responses in PLN cell cultures derived from TNP-Ficoll-primed animals compared to saline control mice. This increased antibody response was TNP-specific as 93% of the PFC's were inhibited by TNP-lysine. Limiting dilution experiments confirm that the increase in anti-TNP PFC response from the TNP-Ficoll-primed animals was indeed due to an increase in TNP-specific precursor B cells. Further, the addition of rIL-5 or rIL-6 induced anti-TNP PFC in the TNP-Ficoll-primed and in control PLN cell cultures in the presence of antigen. However, in primed PLN cells lymphokines alone were sufficient to restore anti-TNP PFC response. In conclusion, our results show that in PLN, the TNP-Ficoll can induce proliferation of hapten-specific B cells but not final differentiation. These primed PLN B cells mature into antibody-secreting cells upon stimulation with TNP-BA or lymphokines.

Animals↗

PubMed1975

DNP-Lys-ficoll: a T-independent antigen which elicits both IgM and IgG anti-DNP antibody-secreting cells.

The 2,4-dinitrophenyl-lysyl derivative of Ficoll (DNP-Lys-Ficoll) was prepared and examined for immunogenicity. This antigen elicited large numbers of DNP-specific plaque-forming cells (PFC) of the IgM and IgG2 class in the spleens of C57BL/6 mice. Similar responses were observed in congenitally athymic (nu/nu) mice and in their littermates indicating that DNP-Lys-Ficoll is a T-independent antigen. The responses of nu/nu mice included a large number of IgG2 DNP-specific PFC, indicating that IgG responses can be initiated in the absence of mature thymus-dependent (T) lymphocytes. Cell transfer studies confirmed the T independence of the response and indicated that priming with DNP-Lys-Ficoll induces only a very meager degree of memory. Because they can be obtained in large quantities and in relatively pure form, DNP-Lys-Ficoll and other hapten conjugates of Ficoll should prove most valuable in the delineation of the mode of activation of precursors of antibody-secreting cells by T-independent antigens.

Animals↗

PubMed1979

Selection of nonmotile Tetrahymena with Ficoll underlayers.

The mechanisms regulating the development of cilia in Tetrahymena are poorly understood but might be revealed through the study of ciliogenesis mutants. Failure to regenerate cilia after dibucaine deciliation results in continued absence of motility. Therefore, to isolate ciliogenesis mutants efficiently, methods for separating motile and nonmotile cells are essential. We examined the efficacy of Ficoll underlayers for these separations. Ciliates of T. thng type IV) were mixed with Ficoll and added as underlayers to separatory funnels containing growth medium. At 27 C most of the cells remained motile and were found in the top layer; at 37 C, there was a time-dependent increase in the number of nonmotile cells and the number of cells in the Ficoll layer. After 150 min at 37 C, most of the cells became nonmotile and were found in the Ficoll layer. Other studies indicated that at 37 C, the cells remained alive and capable of regenerating cilia when deciliated. Thus, it is clear that the Ficoll underlayer effectively separates the majority of nonmotile cells from the majority of motile cells. Evidently, however, at 37 C wild-type T. thermophila exhibit temperature-sensitive phenotypic variability with regard to motility which should be minimized when selecting for mutations affecting motility and ciliogenesis.

Animals↗

PubMed1976

Immunological memory after priming with a thymus independent antigen, NIP-ficoll. 4-hydroxy-5-iodo-3-nitrophenylacetyl coupled to polymer of sucrose and epichlorhydrin.

The capacity of mouse spleen fragments to mount an anti-NIP (4-hydroxy-5-iodo-3-nitrophenylacetyl) response in vitro was studied. The fragments came from unprimed, NIP-Ficoll (polymer of sucrose and epichlorhydrin) or NIP-CG (chicken globulin) primed mice. Unprimed spleen fragments from C57BL/6 mice gave a good anti-NIP response to NIP-Ficoll, whereas CBA fragments did not. Priming with NIP-Ficoll made CBA fragments responsive and enhanced slightly the response of C57BL/6 fragments when stimulated with the same antigen. This memory effect could be seen only after a small priming dose. Priming the mice with NIP-Ficoll made their spleen fragments responsive to a protein conjugate of NIP (NIP-CG), but this effect was seen only after priming with a high dose. The antibody class distribution and the kinetics of the appearance of different immunoglobulin classes were similar in the primary and secondary responses in vitro. The peak responses of IgM, IgA and IgG were reached on day eight and the relative amount of IgG was the same in the primary and in the secondary responses. Spleen fragments derived from NIP-CG primed mice produced more IgG anti-NIP antibodies than fragments derived from untreated mice when immunized in vitro with NIP-Ficoll. The amount of IgG was, however, much higher when these fragments were challenged with the homologous antigen, NIP-CG.

Animals↗

PubMed1978

Comparison of the densities of clonogenic cells from EMT6 fibrosarcoma monolayer cultures, multicell spheroids, and solid tumors in ficoll density gradients.

The densities of viable EMT6 cells grown in vitro as monolayer exponential and plateau-phase cells or as multicell spheroids or in vivo as a solid tumor in BALB/c mice were determined with the use of isopyknic centrifugation in linear Ficoll gradients. Exponential cells banded at a density of 1.069 g/ml, whereas plateau-phase cells appeared at 1.073 g/ml. Cells grown as spheroids were more dense than the monolayer cells and were recovered mainly at 1.081. Solid-tumor cells, separated under the same conditions, banded at 1.080. Narrowing the range of the Ficoll gradient failed to resolve more than one band of cells in the solid-tumor separation. This provides evidence that the density of cells obtained from the spheroids is greater than that of the monolayer cells but agrees well with the density of the solid-tumor cells. Ficoll was demonstrated to be nontoxic to the cells, and plating efficiency assays showed similar cell viabilities between noncentrifuged and centrifuged cells. The plating efficiency of the peak fraction of exponential cells after centrifugation was 68%, and that of plateau-phase cells was 50%. Corresponding figures for the multicell spheroid and solid-tumor cells were 62 and 28%, respectively. The recovery of cells after centrifugation in the Ficoll gradients ranged from 62 to 83%. The effects of cell load and centrifugation time on the density distributions of the EMT6 cells were also investigated.

Animals↗

PubMed1979

Comparison of lymphocyte function after isolation by ficoll-hypaque flotation or elutriation.

Mononuclear leukocytes were separated from whole blood by ficoll-hypaque flotation and by elutriation (counterflow centrifugation). Lymphocytes isolated from 6 control subjects by elutriation showed a 30% greater response to stimulation with phytohemagglutinin and 130% greater response to streptokinase-streptodornase stimulation than did autologous lymphocytes obtained by ficoll-hypaque separation. Cell yields of major mononuclear cell subpopulations and cell viability were comparable after separation of leukocytes by both techniques. These results indicate that ficoll-hypaque flotation may diminish lymphocyte responses, and that elutriation offers a useful alternative to ficoll-hypaque separation. In addition, elutriation may be the preferable method for evaluation of lymphocytes from patients with suspected immunologic dysfunction and may be valuable in the isolation of mononuclear cells from infiltrated skin lesions.

Cell Separation↗

PubMed1979

Simple method for the collection of pancreatic islets by the use of Ficoll-Conray gradient.

Simple method for the collection of large numbers of viable pancreatic islets from normal rats is described. This method involves collagenase digestion of the pancreas, followed by the use of Ficoll-Conray discontinuous gradient for the separation of isolated islets from unwanted acinal debris. Ficoll-Conray A solution was prepared by mixing undialyzed 12.5% Ficoll and 33.4% Conray in the ratio 2 : 1, to make a specific gravity of 1.095 and osmolarity of 396 mOsm/l. The solution B, C, and D, with specific gravities 1.084, 1.072, and 1.048, respectively were obtained by diluting A solution with distilled water. Using Ficoll-Conray gradient, about 200 viable islets which maintained excellently their morphology and function, were collected consistently from a young adult rat pancreas.

Animals↗

PubMed1975

Thymus-independent antigens: the preparation of covalent, hapten-ficoll conjugates.

A general approach is presented for covalently attaching carboxyl and primary amino groups to Ficoll and other high molecular weight polysaccharides in a readily controlled manner. The polysaccharides are omicron-carboxymethylated with chloroacetate in NaOH solutions. Amino groups are next introduced by monoamide formation with ethylenediamine using a water-soluble carbodiimide. Reactions are carried out in aqueous solutions; excess reactants and by-products are removed by dialysis. Three different ways for coupling the 2,4-dinitrophenyl hapten to the amino derivative of Ficoll are described. Preliminary evidence indicates that 2,4-dinitrophenyl-Ficolls, prepared by procedures developed in this study, are potent thymus-independent antigens which can specifically stimulate B cells both in vivo and in vitro. Hapten-Ficoll conjugates should be exceptionally useful for studies of B lymphocyte activation because of the ready availability of the starting material and the ease with which a wide variety of hapten derivatives may be prepared.

Amino Sugars↗

PubMed1978

DNA synthesis and thymidine kinase activity of rat colon epithelial cells fractionated by discontinuous Ficoll gradient.

Epithelial cells from colons of adult Sprague-Dawley rats were fractionated on a discontinuous Ficoll gradient at low centrifugal forces (170 x g) for approximately 60 minutes. Epithelial cells were separated into three distinct zones, whereas cell debris, yeast, and bacteria remained at the top of the gradient. The percentage of cells in each zone was inversely related to the density of the gradient. More than 95% of the cells were morphologically intact and viable (excluded trypan blue). Cells sedimenting at higher densities of Ficoll exhibited higher thymidine kinase activity and DNA synthesis, suggestive of active cell division. The cells sedimenting at lower densities of Ficoll showed the least thymidine kinase activity and DNA synthesis, properties that are compatible with those of mature absorptive cells. Tall columnar cells with vesicular nuclei were predominant in the fraction sedimenting at the lowest density (top fraction). At higher densities (middle and lower fractions), most of the cells were short and columnar with basally located condensed dark-staining nuclei.

Animals↗

PubMed1990

The evaluation of neutral density separation utilizing Ficoll-sodium diatrizoate and Nycodenz and centrifugal elutriation in the purification of bovine and canine islet preparations.

This study evaluates the potential of a neutral density separation technique using iodinated gradient media in combination with Ficoll or alone at two different densities for the purification of bovine and canine islets isolated by a continuous digestion-filtration technique. In addition, centrifugal elutriation is applied to the purification of isolated bovine islets. A single density layer of Ficoll-sodium diatrizoate or Nycodenz at densities of 1.080 g/ml or 1.085 g/ml resulted in an recovery of 47.4% to 77.4% bovine islet volume with bovine islet purities ranging from 43.5% to 49.0%. The use of a single density layer of Ficoll-sodium diatrizoate at 1.080 g/ml and Nycodenz at 1.085 g/ml resulted in recoveries of 86.4% and 56.2% canine islet volume with canine islet purities of 58% and 63.5%, respectively. The bovine islet enriched fraction (flow rates 50-90 ml/min at a rotor speed of 200 rpm) obtained by centrifugal elutriation was 20.0 +/- 3.5% pure, the average recovery of bovine islet volume obtained by centrifugal elutriation was 59.0 +/- 10.7%. It is suggest that a further refinement of factors like osmolality, viscosity, pH, ionic composition and temperature of iodinated density gradient media may allow continued improvement of islet purity and recovery obtained by the simple neutral density separation technique.

Animals↗

PubMed1990

Simple and rapid isolation of platelets using Ficoll-Uropoline cushion.

A simplified procedure for separation of human platelets from plasma proteins when a small volume of blood is available has been developed. In this method two centrifugations of platelets layered on Ficoll-Uropoline cushion was applied. In the first step, platelet rich plasma layered over Ficoll-Uropoline solution was centrifuged (700 g, 30 min), bounded platelets recovered with the help of HEPES buffer, and centrifuged again (1000 g, 10 min) on Ficoll-Uropoline cushion. This rapid and simple procedure yielded platelets separated from von Willebrand factor, fibrinogen and albumin, as judged by bioassay and measurement of radiolabeled plasma proteins. The obtained preparation of platelets was stable and the platelet function preserved for 3 h as evidenced by platelet aggregation and fibrinogen binding analysis. The procedure is inexpensive and convenient for small volumes of blood.

Blood Platelets↗

PubMed1976

Studies on immune responses to parasite antigens in mice. IV. Inhibition of an anti-DNP antibody response with the antigen, DNP-Ficoll containing phosphorylcholine.

Several nematode parasites contain phosphorylcholine (PC), and mice infected with Ascaris suum and Nippostrongylus brasiliensis produce IgM anti-PC antibodies. PC is a hapten which usually induces highly restricted antibody responses in mice and the conjugate DNP-Ficoll-PC inhibits an adoptive secondary anti DNP antibody response to DNP-flagellin whereas comparable doses of DNP-Ficoll and Ficoll-PC do not. The finding suggests that the restricted anti-PC responses to PC-containing antigens described in the literature may involve a degree of inhibition within the B-cell population (partial tolerance induction), that uni- and multi-cellular parasites of various types may 'utilize' molecules such as PC to induce a state of selective tolerance to parasite antigens, and that such a mechanism may be one means of facilitating survival of the parasites in their hosts and the production of antigenic variants of the parasite.

Animals↗

PubMed1978

Purification of human monocytes by continuous gradient sedimentation in ficoll.

A new method of obtaining purified human monocytes has been developed. The peripheral blood mononuclear leukocytes are isolated by centrifugation over Ficoll--Hypaque and then further purified by sedimentation over a linear 5--10% Ficoll density gradient. In ten experiments, the average purity obtained was 77.1% macrophages and the mean yield was 22.4% of the monocytes contained in the peripheral blood leukocytes. Viability of monocytes isolated by this technique exceeded 95%. The cells were phagocytic and responded to human migration inhibitory factor.

Blood Cell Count↗

PubMed1979

Colloidal silica--aluminum modified--PVP density gradient centrifugation: centrifuge tube wall cell adherence, aggregation, separation properties and comparison to BSA and Ficoll.

A method is described for the inexpensive and easy preparation of colloidal silica--aluminum modified--polyvinylpyrrolidone (CS-AM-PVP) density gradient centrifugation medium. Using density gradient centrifugation, several cell separation and biochemical characteristics were studied: centrifuge tube wall cell adherence, mouse spleen cell density distribution, rebanding properties, mitogen response and cell aggregation. Cell adherence to the centrifuge tube wall using density gradients of CS-AM-PVP was compared with density gradients of bovine serum albumin and Ficoll. Few cells adhered to the centrifuge tube wall when CS-AM-PVP was used as a gradient medium; whereas, significant cell adherence to the centrifuge tube cell wall occurred when bovine serum albumin or Ficoll was used as a gradient medium. The CS-AM-PVP gradient medium did not inhibit the response to mitogens of mouse spleen cells which had been separated into density subpopulations in a discontinuous CS-AM-PVP density gradient, caused a minimum amount of cell aggregation, and was found to be non-toxic.

Aluminum↗

PubMed1992

Lymphocyte immunophenotyping by flow cytometry in normal adults. Comparison of fresh whole blood lysis technique, Ficoll-Paque separation and cryopreservation.

In the present report we have assessed the extent to which Ficoll-Paque separation and cryopreservation of mononuclear cells alter the measurement of lymphocyte subsets by flow cytometry. Standard Ficoll-Paque separation increased the percentage of CD4+, CD19+ and CD4+CD45RA+ cells, as well as decreasing that of CD8+, and CD4+CD29+ cells, compared to the fresh whole blood lysis technique. Moreover, cryopreservation caused a depletion of CD4+ p80+ cells, but normal whole blood values were restored following a short incubation.

Adult↗