Characterization of the separation properties of the Beckman elutriator system

The role of fluid flow in the elutriation process was visualized by pumping dye solution through the Beckman JE-6 elutriator rotor. Three major fluid flow disturbances were observed in the separation chambers, namely; jet-streaming, ripple flow, and whirl flow. In order to evaluate the effects of these non-ideal fluid flow patterns on the separation of homogeneous populations of particles or cells, 12--35 micron diameter latex spheres and 9L rat brain tumor cells were fractionated with the Beckman elutriator system. The elutriator system was evaluated on the basis of: (1) recovery, (2) elution loss during loading, (3) homogeneity of the size distributions, and (4) the relationship of the median volume of eluted particles or cells to the rotor speed and the collection fluid velocity. Both a conventional collection method (two 40-mL fractions at ech collection rotor speed) and a long collection method (10--15 40-mL fractions at several collection rotor speeds) were compared to determine if collection procedures could compensate for some of the difficulties caused by the non-ideal fluid flow patterns. Although more than 90% of the particles or cells were always recovered, about 5% eluted during the loading procedure. Neither collection method altered this phenomenon. The long collection method significantly improved the homogeneity of the collected populations, but this was accompanied by a reduction in cell yield. The median particle or cell volume of each fraction agreed with that expected under ideal fluid flow conditions except at high and low rotor speeds when the conventional collection method was used.     

Resolution of cells by centrifugal elutriation

Cell separation using the Beckman elutriator depends upon the flow rate of the medium and the centrifugal field employed. Changes in either the centrifugal field or the flow rate can be used to elute fractions of cells based on size. Even when these variables are held constant in the Beckman J21C centrifuge, a periodic pulse of cells is eluted. We have found that this anomolous elution is related to the temperature control system which gave a periodically pulsed temperature drop in the centrifuge well. The elution resulting from this change in temperature caused a shift in the modal cell size of the fraction eluted at a particular flow rate and centrifugal field. Because of this, the fractions have a larger size dispersion than fractions collected under conditions where refrigeration-related temperature fluctuations do not occur. We conclude that the temperature control system of the Beckman J21C centrifuge used with the Beckman elutriation rotor produces temperature fluctuations which prevent maximum resolution of cells.     

One-step isolation of neutrophils using an elutriator

Summary To simplify the isolation of neutrophils, we developed a one-step procedure using elutriation. The perfusate (0.2% gelatin and 0.1% glucose in phosphate buffered saline) was pumped through an elutriator rotor at 4 ml/min (25° C) with the rotor speed at 2370 rpm. Twenty milliliters of anticoagulated porcine venous blood were mixed with 60 ml of perfusate and loaded into the elutriator chamber. The flow rate was increased by 2 ml/min increments and 100-ml fractions of effluent were collected at each increment. Concentrations of neutrophils and mononuclear cells were measured in each fraction, and the percentage of total neutrophils or mononuclear cells was plotted against flow rate. The optimal yield (46%) and purity (95.1%) of neutrophils (n=8) was obtained in pooled fractions at flow rates greater than 20 ml/min. Neutrophils in this preparation were round, the granules were intact, and the nuclei were lobulated. In addition, the cells produced superoxide in the presence of phorbol myristate acetate and phagocytosed zymosan particles. These characteristics were similar to those of porcine neutrophils prepared by a conventional sedimentation method. The yield (43%) and purity (94%) of human neutrophils isolated using the elutriator method was similar to that for porcine cells. This one-step method provides a moderate yield of pure neutrophils that have retained their morphology and function. This work was supported by the Canadian Heart Foundation.     

The use of counterflow centrifugation to enrich gonadotropes and somatotropes.

Counterflow centrifugation produces populations of gonadotropes or growth hormone (GH) cells enriched to 90% in a Beckman elutriator. The pituitary populations are first separated by size into three fractions applying different flow rates, stimulated with either gonadotropin-releasing hormone (GnRH) to enlarge the gonadotropes or growth hormone-releasing hormone (GHRH) to enlarge the somatotropes for 3 hr. The fractions are re-eluted, first at the original flow rates and then at higher flow rates to separate enlarged gonadotropes or somatotropes. Most other cell types are reduced to less than 5%. However, co-storage of GH and gonadotropin antigens is seen in either population. Enriched gonadotropes or somatotropes can be used in studies of proliferation, autocrine or paracrine regulation, or ion channel functions.(J Histochem Cytochem 49:663-664, 2001)     

Isolation and identification of clara cells from rabbit lung

Summary A procedure has been developed for the isolation of nonciliated bronchiolar epithelial cells (Clara cells) from rabbit lung. Following pulmonary lavage to eliminate macrosphages, cells (5% Clara cells) were released by digestion with 0.1% Protease I in HEPES-buffered balanced salt solution containing 0.5 mM ethylene glycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid instilled through the trachea. These cells were then separated on the basis of size using the Beckman JE-6 elutriator rotor. The fourth fraction collected from the elutriator contained about 30% Clara cells. This fraction was then layered on a two-polymer aqueous phase system consisting of 5% dextran T500 (DT) and 3.8% polyethylene glycol 6000 (PEG) in sodium phosphate buffer. A cell fraction was obtained from the PEG phase, which included approximately 70% Clara cells. These cells were found to be greater than 90% viable by trypan blue dye exclusion. Identification of isolated Clara cells was confirmed by light microscopic observation of nitro blue tetrazolium staining and by ultrastructural characteristics as observed by electron microscopy. This research was supported in part by the U.S. Environmental Protection Agency under an interagency agreement relating to the Federal Interagency Energy/Environmental Research and Development Program.     

Synchronization of 9L rat brain tumor cells by centrifugal elutriation

Asynchronous 9L cells were separated into relatively homogeneously-sized populations using centrifugal elutriation with both a conventional collection method and a long collection method. A substantial increase in the homogeneity of the volume distributions and in the degree of synchrony of the separated fractions was obtained using the long collection method. Autoradiographic data indicated that fractions containing ≥97% G₁ cells, ≥80% S cells, and 70–75% G₂ cells could be routinely recovered with this procedure. Recovery in these fractions varied from 5 to 8% of the total number of cells elutriated. The colony forming efficiency (CFE) of cells from fractions representing each phase of the cell cycle was a constant 60–70%, which was comparable to the 60–80% usually found for asynchronous 9L cells. The percentage of cells in the G₁, S, and G₂ phases in the elutriated fractions was more accurately determined from the volume distribution than from computer fits of the DNA histogram obtained from flow cytometry. In general, the degree of synchrony was related to the coefficient of variation (CV) of the volume distributions of the elutriated fractions. The CV was about 14% for all elutriated fractions. When the ≥97% G₁ population was allowed to progress to S and G₂, the CVs were about 17 and 20.2%, respectively. Thus, the best nonperturbing method for obtaining synchronous 9L cells in the S or G₂ phases was direct elutriation with the long collection method.     

Embedding Free-Floating Cells and Microscopic Particles: Serum Albumin Coagulum—Epoxy Resin

Small quantities of cells or microscopic particles are fixed in suspension, centrifuged at low speed in a conical tube and the supernatant fluid removed as completely as possible. The pellet is resuspended in 0.25 ml of 2% bovine serum albumin (in 0.05 M Tris buffer, pH 7.5) and transferred to a small, conical cellulose tube (Beckman #303369). One drop of 25% glutaraldehyde is mixed with the suspension and the tube is quickly centrifuged in a swinging bucket rotor. A gel forms in about 5 min and the coagulum can be cut and processed through embedding like small blocks of tissue.     

Bulk Separations of Rat Brain Cells by Centrifugal Elutriation

Six day old rat cerebrums prepared as a 10% cell suspension in 3% buffered Ficoll medium were fractionated in a Beckman CR-3 Elutriator rotor system. Neuroglial cells and heterogeneous components of broken cells were separated from intact neuronal perikarya at rotor speeds, respectively, of 2400 rev/min (first pass) and 5030 rev/min (second pass).     

Classification and Phylogenetic Analysis of the cAMP-Dependent Protein Kinase Regulatory Subunit Family

The members of the PKA regulatory subunit family (PKA-R family) were analyzed by multiple sequence alignment and clustering based on phylogenetic tree construction. According to the phylogenetic trees generated from multiple sequence alignment of the complete sequences, the PKA-R family was divided into four subfamilies (types I to IV). Members of each subfamily were exclusively from animals (types I and II), fungi (type III), and alveolates (type IV). Application of the same methodology to the cAMP-binding domains, and subsequently to the region delimited by β-strands 6 and 7 of the crystal structures of bovine RIα and rat RIIβ (the phosphate-binding cassette; PBC), proved that this highly conserved region was enough to classify unequivocally the members of the PKA-R family. A single signature sequence, F–G–E–[LIV]–A–L–[LIMV]–x(3)–[PV]–R–[ANQV]–A, corresponding to the PBC was identified which is characteristic of the PKA-R family and is sufficient to distinguish it from other members of the cyclic nucleotide-binding protein superfamily. Specific determinants for the A and B domains of each R-subunit type were also identified. Conserved residues defining the signature motif are important for interaction with cAMP or for positioning the residues that directly interact with cAMP. Conversely, residues that define subfamilies or domain types are not conserved and are mostly located on the loop that connects α-helix B′ and β strand 7. Received: 2 November 2000/Accepted: 14 June 2001     

Seasonal changes in fatty acid composition associated with cold-hardening in third instar larvae of Eurosta solidaginis

Third-instar larvae of the goldenrod gall fly Eurosta solidaginis (Diptera: Tephritidae) survive extended periods in winter during which tissue water is frozen. Both low temperature and reduced water activity during freezing present challenges for the structural integrity of cellular lipids. Fatty acids of both phospholipids and triacylglycerols from fat body cells of E. solidaginis were analyzed throughout fall and early winter, a period that encompasses the acquisition of freeze-tolerance, to determine if adaptations to freezing include changes in fatty acid unsaturation. The five most abundant fatty acids from both fractions were (in decreasing order) oleic (40–65%), palmitoleic (18–20%), palmitic (12–17%), linoleic (5–10%), and stearic acids (4 –7%). This represents a typical complement of Dipteran fatty acids, although oleic acid levels were higher in E. solidaginis than those reported from other Dipterans (˜28%; Downer 1985). From September to November, monounsaturates increased from 59 to 70% in phospholipids at the expense of saturated fatty acids (25% –20%) suggesting activation of a Δ⁹-desaturase enzyme. These changes resulted in an increase in the ratio of unsaturated to saturated fatty acids (U/S) from 3.0 to 4.2, although there was no change in the average number of double bonds per fatty acid (unsaturation index, UI ≈ 1.2 in phospholipids and 0.9 in triacylglycerols throughout the season). These changes were temporally correlated to decreasing ambient temperatures and increasing larval and fat body cell freeze-tolerance. Accepted: 31 October 1996     

The influence of either no fluid or carbohydrate-electrolyte fluid ingestion and the environment (thermoneutral versus hot and humid) on running economy after prolonged, high-intensity exercise

This study investigated the effects on running economy (RE) of ingesting either no fluid or an electrolyte solution with or without 6% carbohydrate (counterbalanced design) during 60-min running bouts at 80% maximal oxygen consumption (V˙O_2max). Tests were undertaken in either a thermoneutral (22–23°C; 56–62% relative humidity, RH) or a hot and humid natural environment (Singapore: 25–35°C; 66–77% RH). The subjects were 15 young adult male Singaporeans [V˙O_2max = 55.5 (4.4 SD) ml kg⁻¹ min⁻¹]. The RE was measured at 3 m s⁻¹ [65 (6)% V˙O_2max] before (RE1) and after each prolonged run (RE2). Fluids were administered every 2 min, at an individual rate determined from prior tests, to maintain body mass (group mean = 17.4 ml min⁻¹). The V˙O₂ during RE2 was higher (P < 0.05) than that during the RE1 test for all treatments, with no differences between treatments (ANOVA). The mean increase in V˙O₂ from RE1 to RE2 ranged from 3.4 to 4.7 ml kg⁻¹ min⁻¹ across treatments. In conclusion, the deterioration in RE at 3 m s⁻¹ (65% V˙O_2max) after 60 min of running at 80% V˙O_2max appears to occur independently of whether fluid is ingested and regardless of whether the fluid contains carbohydrates or electrolytes, in both a thermoneutral and in a hot, humid environment. Accepted: 30 October 1997     

High gradient magnetic separation of rosette-forming cells

High Gradient Magnetic Separation (HGMS) is a rapid and straightforward technique that has previously been proven effective in extracting erythrocytes from a flowing cell suspension if the red cell hemoglobin is in a paramagnetic state. In this work it was applied to the enrichment of the small population (<2%) of splenocytes from an immune mouse that bound sheep red cells to form rosettes. Samples flowed through the HGMS column in a strong magnetic field where rosettes and free sheep cells were selectively retained. These were subsequently eluted by simply removing the magnetic field. The process required 20–30 min per mouse spleen. Rosettes in the initial sample and in the fractions that passed through, or were retained by, the column were enumerated under the microscope. Under the conditions used here, the retained and eluted cells typically showed a 20–50-fold increase in the frequency of rosetted cells, and the cells that passed through the magnet showed 90–100% depletion of rosettes. The recovery of intact rosettes and the overall cell recovery were generally both in the range of 80–90%.     

Effect of Antipeptide Antibodies Directed against Three Domains of Connexin43 on the Gap Junctional Permeability of Cultured Heart Cells

Cell-to-cell communication can be blocked by intracellular injections of antibodies raised against gap junction proteins, but the mechanism of channel obstruction is unknown. Binding to connexins could lead to a conformational change, interfere with regulatory domains or cause a steric hindrance. To address these questions, the effects on cell-to-cell communication of affinity purified polyclonal antibodies raised against peptides reproducing the intracellular sequences 5–17, 314–322 and 363–382 of rat connexin43 were investigated in cultured rat ventricular cells. The antibodies against sequence 363–382 were characterized by immunoblotting and immunocytochemistry. Characterization of antibodies 5–17 and 314–322 has been previously reported. In a first series of experiments, the effect on gap junctional communication was assessed by injecting a junction-permeant fluorescent dye into cells adjacent to one cell previously microinjected with antibodies. In a second series, junctional permeability was quantitatively determined on records of fluorescence recovery after the photobleaching of 6-carboxyfluorescein-loaded cells. Antibodies 5–17 marked a 43 kDa band on immunoblots, but did not immunolabel gap junctions and had no functional effect. Antibodies 314–322 recognized the 43 kDa protein and labeled the intercalated disks, but failed to interfere with junctional permeability. Antibodies to the nearby sequence 363–382, for which all immunospecific tests had been positive, caused a delayed diffusional uncoupling in 50% of the microinjected cells. It is suggested that the blocking of junctional communication by antibodies results from interference with a regulatory domain of the connexin. Received: 25 July 1995/Revised: 21 December 1995     

CFTR Activation Raises Extracellular pH of NIH/3T3 Mouse Fibroblasts and C127 Epithelial Cells

Cystic Fibrosis (CF) is caused by mutations in the gene for CFTR, a cAMP-activated anion channel found in apical membranes of wet epithelia. Since CFTR is permeable to HCO⁻ ₃ and changes in extracellular fluid composition may contribute to CF lung disease, we investigated possible differences in extracellular pH (pHo) between CFTR-expressing and control cell lines. The Cytosensor™ Microphysiometer was used to study forskolin-stimulated extracellular acidification rates in CFTR-expressing and control mouse mammary epithelial (C127) and fibroblast (NIH/3T3) cell lines. Forskolin, which activates CFTR via raised cAMP, caused decreased extracellular acidification of CFTR-expressing NIH/3T3 and C127 cells by 15–35%. By contrast, forskolin caused increased extracellular acidification of control cells by 10–20%. Ionomycin, which may activate CFTR via PKC, also elicited this decreased extracellular acidification signal only in cells expressing CFTR. In control experiments, dideoxyforskolin had no effect on the acidification rates and osmotic stimuli were shown to equally stimulate all cell lines. These results suggest a role for CFTR in controlling pHo and complement recent evidence that HCO⁻ ₃ dependent epithelial secretion may be reduced in amount and altered in composition in CF. Received: 20 June 2000/Revised: 13 November 2000     

Three-dimensional finite element modeling of pericellular matrix and cell mechanics in the nucleus pulposus of the intervertebral disk based on in situ morphology

Nucleus pulposus (NP) cells of the intervertebral disk (IVD) have unique morphological characteristics and biologic responses to mechanical stimuli that may regulate maintenance and health of the IVD. NP cells reside as single cell, paired or multiple cells in a contiguous pericellular matrix (PCM), whose structure and properties may significantly influence cell and extracellular matrix mechanics. In this study, a computational model was developed to predict the stress–strain, fluid pressure and flow fields for cells and their surrounding PCM in the NP using three-dimensional (3D) finite element models based on the in situ morphology of cell–PCM regions of the mature rat NP, measured using confocal microscopy. Three-dimensional geometries of the extracellular matrix and representative cell–matrix units were used to construct 3D finite element models of the structures as isotropic and biphasic materials. In response to compressive strain of the extracellular matrix, NP cells and PCM regions were predicted to experience volumetric strains that were 1.9–3.7 and 1.4–2.1 times greater than the extracellular matrix, respectively. Volumetric and deviatoric strain concentrations were generally found at the cell/PCM interface, while von Mises stress concentrations were associated with the PCM/extracellular matrix interface. Cell–matrix units containing greater cell numbers were associated with higher peak cell strains and lower rates of fluid pressurization upon loading. These studies provide new model predictions for micromechanics of NP cells that can contribute to an understanding of mechanotransduction in the IVD and its changes with aging and degeneration.     

Reversible Electropermeabilization of Mammalian Cells by High-Intensity, Ultra-Short Pulses of Submicrosecond Duration

Mouse myeloma cells were electropermeabilized by single square-wave electric pulses with amplitudes of up to ∼150 kV/cm and durations of 10–100 nsec. The effects of the field intensity, pulse duration and medium conductivity on cell viability and field-induced uptake of molecules were analyzed by quantitative flow cytometry using the membrane-impermeable fluorescent dye propidium iodide as indicator molecule. Despite the extremely large field strengths, the majority of cells survived the exposure to ultra-short field pulses. The electrically induced dye uptake increased markedly with decreasing conductivity of the suspending medium. We assigned this phenomenon to the transient electrodeformation (stretching) force that assumes its maximum value if cells are suspended in low-conductivity media, i.e., if the external conductivity σ_e is smaller than that of the cytosol σ_i. The stretching force vanishes when σ_e is equal to or larger than σ_i. Due to their capability of delivering extremely large electric fields, the pulse power systems used here appear to be a promising tool for the electropermeabilization of very small cells and vesicles (including intracellular organelles, liposomes, etc.). Received: 15 May 2001/Revised: 20 July 2001     

Evaluation of 28 marine algae from the Qingdao coast for antioxidative capacity and determination of antioxidant efficiency and total phenolic content of fractions and subfractions derived from Symphyocladia latiuscula (Rhodomelaceae)

The extracts obtained from 28 species of marine algae were evaluated for their antioxidant activity (AA) versus the positive controls butylated hydroxytoluene (BHT), gallic acid (GA), and ascorbic acid (AscA). Most of the tested samples displayed antioxidant activity to various degrees. Among them, the extract of Symphyocladia latiuscula exhibited the strongest AA, which was comparable to BHT, GA, and AscA in radical scavenging activity, as shown in the DPPH (α,α-diphenyl-β-picrylhydrazyl) assay, and higher than those of the positive controls in β-carotene-linoleate assay system. In addition, the ethyl acetate-soluble fraction isolated from the crude extract of S. latiuscula exhibited the highest antioxidant activity in both assay systems. This fraction was further fractionated into seven subfractions (F1-F7) by vacuum liquid chromatography (VLC). F1 and F4 were found to be the most effective subfractions in scavenging DPPH radical assay and in the β-carotene-linoleate assay, respectively. The total phenolic content (TPC) and reducing power (RP) for all of the extracts, fractions, and subfractions (F1–F7) were also determined. The TPC of the 28 extracts ranged from 0.10 to 8.00 gallic acid equivalents (mg/g seaweed dry weight) while the RP ranged from 0.07 to 11.60 ascorbic acid equivalents (mg·g⁻¹ seaweed dry weight). Highly positive relationships between AA and TPC as well as between AA and RP were found for the extracts and fractions, while for the subfractions F1–F7 only weak or no such relations were found. The results obtained from this study indicate that further analysis is needed of those marine algal species that contain the most antioxidant activity in order to identify the active principles.     

Intracellular chloroplast photorelocation in the moss Physcomitrella patens is mediated by phytochrome as well as by a blue-light receptor

 The light-induced intracellular relocation of chloroplasts was examined in red-light-grown protonemal cells of the moss Physcomitrella patens. When irradiated with polarized red or blue light, chloroplast distribution in the cell depended upon the direction of the electrical vector (E-vector) in both light qualities. When the E-vector was parallel to the cross-wall (i.e. perpendicular to the protonemal axis), chloroplasts accumulated along the cross-wall; however, no accumulation along the cross-wall was observed when the E-vector was perpendicular to it (i.e. parallel to the protonemal axis). When a part of the cell was irradiated with a microbeam of red or blue light, chloroplasts accumulated at or avoided the illumination point depending on the fluence rate used. Red light of 0.1–18 W m⁻² and blue light of 0.01–85.5 W m⁻² induced an accumulation response (low-fluence-rate response; LFR), while an avoidance response (high-fluence-rate response; HFR) was induced by red light of 60 W m⁻² or higher and by blue light of 285 W m⁻². The red-light-induced LFR and HFR were nullified by a simultaneous background irradiation of far-red light, whereas the blue-light-induced LFR and HFR were not affected at all by this treatment. These results show, for the first time, that dichroic phytochrome, as well as the dichroic blue-light receptor, is involved in the chloroplast relocation movement in these bryophyte cells. Further, the phytochrome-mediated responses but not the blue-light responses were revealed to be lost when red-light-grown cells were cultured under white light for 2 d. Received: 7 September 1999 / Accepted: 15 October 1999     

Two modes of sulfite oxidation in the extremely thermophilic and acidophilic archaeon Acidianus ambivalens

Sulfite-oxidizing enzyme activities were analyzed in cell-free extracts of aerobically grown cells of Acidianus ambivalens, an extremely thermophilic and chemolithoautotrophic archaeon. In the membrane and cytoplasmic fractions, two distinct enzyme activities were found. In the membrane fraction, a sulfite:acceptor oxidoreductase activity was found [530 mU (mg protein)^–1; apparent K _m for sulfite, 3.6 mM]. In the cytoplasmic fraction the following enzyme activities were found and are indicative of an oxidative adenylylsulfate pathway: adenylylsulfate reductase [138 mU (mg protein)^–1], adenylylsulfate:phosphate adenyltransferase [“ADP sulfurylase”; 86 mU (mg protein)^–1], adenylate kinase [650 mU (mg protein)^–1], and rhodanese [thiosulfate sulfur transferase, 9.2 mU (mg protein)^–1]. In addition, 5′,5′′′-P¹,P⁴-di(adenosine-5′) tetraphosphate (Ap₄A) synthase and Ap₄A pyrophosphohydrolase activities were detected. Received: 17 August 1998 / Accepted: 29 April 1999