Volker Kachel

Swelling, Acidosis, and Irreversible Damage of Glial Cells from Exposure to Arachidonic Acid in vitro

Swelling and damage of C6 glioma cells and of primary cultured astrocytes were analyzed in vitro during incubation with arachidonic acid (AA; 20:4). The cells were suspended in a physiological medium supplemented with AA at concentrations of 0.001–1.0 mM. Cell swelling was quantified by flow cytometry with hydrodynamic focusing. Flow cytometry was also utilized for assessment of cell viability by exclusion of the fluorescent dye propidium iodide and for measurement of the intracellular pH (pHi) by 2′,7′-bis-(2-carboxyethyl)−5(and −6)carboxyfluorescein. Administration of AA caused an immediate dose-dependent swelling of C6 glioma cells, even at a concentration of 0.01 mM. At this level cell volume increased within 20 min to 105.0% of control, at 0.1 mM to 111.0%, while at 1.0 mM to 123.7%. Following a phase of rapid cell volume increase, swelling leveled off during the subsequent observation period of 70 min. Viability of the C6 glioma cells was 90% under control conditions. It remained unchanged after raising AA concentrations to 0.1 mM. At 0.5 mM, however, cell viability fell to 72.8%, and at 1.0 mM to 32.7%. pHi of the glioma cells was 7.3 under control conditions. In parallel with the early swelling phase, AA led to a dose-dependent decrease of the intracellular pH and an elevated lactate production of the cells. During incubation with 0.1 mM AA, pHi decreased to 7.06 after 5 min, but recovered to normal subsequently. In addition, swelling-inducing properties of linoleic (18:2) or stearic (18:0) acid were analyzed for evaluation of the specificity of glial swelling induced by AA. Whereas stearic acid (0.1 mM) failed to induce a swelling response, linoleic acid (0.1 mM) was found to be effective. The volume increase of the glial cells, however, was only half of that found during exposure to AA at the same concentration. Further, glial swelling from AA or linoleic acid was completely inhibited by the aminosteroid U-74389F, an antagonist of lipid peroxidation. Finally, omission of Na+ ions in the suspension medium with replacement by choline led also to inhibition of the cell volume increase by AA. Experiments using astrocytes from primary culture confirmed the swelling-inducing properties of AA at a quantitative level, whereas vulnerability of the cells to AA was increased. The present results demonstrate an important role of AA in cytotoxic swelling and irreversible damage of glial cells at concentrations that occur in vivo in cerebral ischemia or trauma. The damaging potential of AA might be enhanced by a concurrently evolving intracellular acidosis, stimulating the formation of oxygen-derived free radicals and lipid peroxidation.

A new multiparameter flow cytometer: optical and electrical cell analysis in combination with video microscopy in flow.

BACKGROUND Flow cytometers, which are commercially available, do not necessarily meet all demands of actual biomedical research. This is the case for the investigation of mechanisms involved in cell volume regulation, which requires electrical volume measurement and ratiometric multichannel fluorescence analysis for the simultaneous assessment of different physiologic parameters (intracellular pH and the intracellular concentration of calcium ions, etc). METHODS AND RESULTS We describe the construction of a new nonsorting flow cytometer designed for the simultaneous acquisition of seven parameters including fluorescence signals, forward and perpendicular light scatter, cell volume according to the electrical Coulter principle, and flow cytometric imaging. The instrument is equipped with three different light sources. A tunable argon-ion laser generates efficient excitation of the most standard fluorescent probes in the visible spectral range, and an arc lamp provides the means for ultraviolet excitation at low cost. Because of the spatial filtering by the excitation and detection optics, two independent sets of dual fluorescence measurements can be performed, a prerequisite for flexible ratiometric fluorescence analysis. A flow video microscope integrated into the optical system optionally generates either brightfield or phase images of selected flowing particles. Only particles whose individual datasets meet predefined gating conditions are imaged in real time. To avoid smear effects, the motion of the object to be imaged (speed approximately 8 m/s) is frozen on the target of a CCD camera by flash illumination. For this purpose, a high radiance gas discharge lamp with 25-mJ electric pulse energy provides an illumination time of 18 ns (full width half maximum). Test results obtained from latex spheres and cells are shown. CONCLUSIONS Test results indicate that our instrument can perform Coulter measurements in combination with flexible optical analysis. Moreover, integration of an adapted video microscope into a flow cytometer is an approach to overcome the gap between flow and image cytometry.

High-throughput isolation of ultra-pure plasmid DNA by a robotic system

BackgroundWith the availability of complete genomes, a systematic inventory of cellular processes becomes achievable. This requires assessing the function of all individual genes. Transfection of plasmid DNA into cell culture cells is an essential technique for this aim as it allows functional overexpression or downregulation of genes. While many robotic systems isolate plasmids for sequencing purposes, for more demanding applications such as transfections there is a shortage of robots for the high-throughput isolation of plasmid DNA.ResultsHere we describe a custom-made, automated device, which uses a special protocol to isolate plasmid DNAs with a purity sufficient for efficient transfections into mammalian cells. Approximately 1,600 ultra pure plasmids can be isolated in a 96-well plate format within 12 hours. As a unique feature the robot comprises the integration of a centrifuge instead of expensive columns, the use of a custom-made pipetting head with a movable gripper, especially designed shaking platforms and an acetone wash facility.ConclusionUsing this robot we demonstrate how centrifugation steps with multiple precipitations, most notably through a precipitation step of SDS in isopropanol, lead to high purity plasmid DNA and make possible high-throughput transfections into mammalian cells for functional gene annotations.

Der Einfluß der Partikeldurchtrittsbahn auf die Volumenverteilungskurven nach dem Coulter-Verfahren

SummaryA measuring chamber is described which functions according to the Coulter principle and with which one can produce adjustable, optically distinguishable particle pathways across the entire measuring orifice. Different pathways through the orifice for particles of a given volume result in different measured values of up to 80% for the volume. The volume distribution for each individual pathway is approximately symmetrical; these individual distributions are weighted by a fluid velocity profile factor and then superimposed to yield a total, right-skewed distribution, theoretically the same as that obtained with an unmodified Coulter capillary where the volume distribution is comprised of all possible pathways. Comparison of the area of this synthetic distribution curve with that of the axial pathway yields a ratio of 1.39, in close agreement with the corresponding ratio of 1.45 for the unmodified Coulter capillary.Particle deformation and rotational alignment during passage through the orifice are also investigated. Forty-nanosecond exposure photographs of native erythrocytes and of fixed erythrocytes in the axial region and near the orifice walls show that (1) cell deformation occurs throughout the entire orifice and (2) an occasional cell near the wall rotates but none in the axial region does so.ZusammenfassungEs wird eine Meßkammer nach dem Coulter Prinzip beschrieben, mit welcher man verstellbare, optisch erfaßbare Teilchenbahnen in der gesamten Meßöffnung erzeugen kann. Mit dieser Kammer wird die unterschiedliche von den Verzerrungen des elektrischen Feldes hervorgerufene Bewertung des Teilchenvolumens gezeigt; die Unterschiede können je nach Teilchenbahn bis 80% betragen. Die weitgehend symmetrischen Verteilungskurven, die einzelnen Bahnen entsprechen, werden unter Berücksichtigung der Geschwindigkeitsverteilung zu einer rechtsschiefen Gesamtverteilung zusammengesetzt. Das Verhältnis der Fläche unter der rechtsschiefen Verteilungskurve zur Fläche der Verteilungskurve der Bahn in der Lochachse beträgt 1,39. Bei vergleichend aufgenommenen Verteilungskurven mit Capillaren mit entfernbarer Zentralstrahlvorrichtung beträgt das Flächenverhältnis von allgemeiner Verteilungskurve zur Zentralstrahlverteilungskurve 1,45. Kurzzeitphotographien zeigen Bahnen von fixierten und nativen Erythrocyten im Meßlochinnern und am Meßlochrand. Dabei wird die Ausrichtung der Zellen in der Strömung und die starke Verformbarkeit der nativen Erythrocyten deutlich.

Der Nachweis verschiedener Erythrozytenpopulationen bei der Ratte

Zusammenfassung1.Auf Grund der Analyse der Volumenverteilungskurven von Rattenerythrozyten lassen sich vom 1. bis 320. Tag nach der Geburt insgesamt 5 normalverteilte, volumenmäßig unterscheidbare Erythrozytenpopulationen nachweisen. Bei der Geburt sind 3 Populationen I bis III vorhanden, die durch linearen Volumenverlust mit der Zeit immer kleiner werden und bei einem kritischen mittleren Volumen von etwa 36μ3 aus der Zirkulation verschwinden. Der Volumenverlust ist sehr wahrscheinlich einer morphologisch nachweisbaren Erythrozytenfragmentierung zuzuschreiben. In der Zeit vom 20. bis 24. Tag nach der Geburt treten 2 neue Populationen IV und V auf, von denen die Population V die Endbevölkerung darstellt, während der Anteil von Population IV um den 84. Tag so gering wird, daß sie nicht mehr mit Sicherheit nachweisbar ist.2.Das Auftreten und Verschwinden mehrerer Erythrozytenpopulationen führt zwischen dem 18. und 31. Tag nach der Geburt zu vorübergehenden Einbrüchen im Kurvenverlauf bei verschiedenen Erythrozytenparametern im Blut, nicht aber zu einer Beeinträchtigung der Gewichtszunahme der Tiere. Daraus wird geschlossen, daß es sich beim Auftreten und Verschwinden der verschiedenen in ihrer Funktion und Herkunft noch weitgehend unbekannten Erythrozytenpopulationen um physiologische Regelungsvorgänge handelt.Summary1.The volume distribution curves of erythrocytes from rats of ages from 1 to 320 days were analysed. Five populations of erythrocytes, possessing different volumes, were observed. All populations showed a Gaussian distribution. The populations I, II and III are present at birth. They show a linear loss in cell volume and disappear from circulation when the mean cellular volume approaches about 36μ3. The volume loss is probably due to erythrocyte fragmentation. From day 20 to 24 after birth, two new populations IV and V appear. Population IV disappears at about 84 days of age; population V is the final erythrocyte population.2.Between 18 to 31 days after birth the appearance and disappearance of several erythrocyte populations in the blood is accompanied by a marked drop in microhematocrit, hemoglobin and eruthrocyte/mm3, the weight increase of the rat, which is a sensitive indicator of an undisturbed growth, is not affected.3.It is concluded that the appearance and disappearance of different rat erythrocyte populations of yet unknown origin and function, reflect physiological regulation mechanisms.

Erythrozytenaggregation bei Nichtrauchern, Rauchern und Herzinfarktpatienten

ZusammenfassungDie auf Grund rheologischer Untersuchungen vermutete erhöhte Aggregationsneigung der Erythrozyten von nicht Kortikoid- oder Colfarit®-behandelten Herzinfarktpatienten konnte experimentell bestätigt werden. Kortikoidgabe oder eine orale Behandlung mit Acetylsalizylsäure (Colfarit®) führen bei Herzinfarktpatienten zu einer beträchtlichen Senkung der Aggregationsneigung. Auch zeigen sonst gesunde starke Raucher gegenüber Normalprobanden eine erhöhte Aggregationsneigung der Erythrozyten.Patienten mit einer beschleunigten Blutkörperchensenkung haben zwar eine gewisse Erhöhung der Erythrozytenaggregation gegenüber Normalpersonen, diese liegt jedoch deutlich unterhalb jener frischer Herzinfarktpatienten.Auf Grund dieser Ergebnisse sollen weitere Risikofaktoren des Herzinfarktes (Diabetes mell., Hypertonie, Adipositas) in ihrer Einwirkung auf den Erythrozyten-Aggregationsindex untersucht werden. Die vorliegenden Ergebnisse geben vielleicht eine Möglichkeit, mit einer schnellen und relativ einfachen Methode frühzeitig eine Herzinfarktgefährdung zu erkennen, sowie eine Erklärung für die erhöhte Infarktgefährdung von starken Rauchern.SummaryIt was supposed by rheological investigations that erythrocytes of non-corticoid or Colfarit® treated infarct patients have an increased aggregation. This could now be confirmed experimentally in comparison to normal persons. Corticoid-treatment or oral treatment with acetylsalicylic-acid (Colfarit®) shows a strong decrease in the aggregation of erythrocytes of infarct patients. Furthermore healthy heavy smokers have an increased aggregation of erythrocytes. Patients with an increased blood sedimentation rate showed also a certain increase of aggregation which however lies beneath that of a just occurred infarct.

The increase of electrophoretic mobility and alkaline phosphatase activity are parallel events during B-cell maturation.

B-lymphocyte derived mouse myeloma cells P3X63Ag8U.1 were used to study the change of the negative surface charge density which occurs during a final maturation step of mouse and human B-cells. These cells showed a uniform EM distribution curve as long as they lived within a clone. However, when they grew in suspension at low density, a part of them increased their electrophoretic mobility (EM). Cells with enhanced EM were isolated by free flow electrophoresis. They showed lower proliferation and clone forming activity but higher alkaline phosphatase activity than cells with unchanged low EM. The study suggests that the increase of the EM and of the alkaline phosphatase activity are parallel events associated with B cell progression from the proliferative to the Ig secretion stage.

The negative surface charge density is a maturation marker of human B lymphocytes

Small resting B lymphocytes were highly enriched and completely depleted of all preactivated large B lymphocytes using countercurrent centrifugal elutriation and free flow electrophoresis. They required T lymphocytes, monocytes, and a mitogen to produce antibodies after 5 days of preincubation. Large activated B lymphocytes were obtained in cell fractions which were free of resting ones. They produced antibodies even in the absence of a mitogen. Two groups were distinguished, differing in their stage of differentiation and their negative surface charge density. The cells of one group had an electrophoretic mobility (EM) like resting B lymphocytes ranging from 0.85 to 0.99 X 10(-4) (cm2 V-1 s-1). They took 2 to 3 days of preincubation before they started to secrete antibodies. Interleukin 2 and pokeweed mitogen enhanced their antibody production capability. The cells of the other group had an EM between 0.99 and 1.13 X 10(-4) (cm2 V-1 s-1). They secreted antibodies even during the first day of incubation. The quantity of the antibodies which they produced depended only on the blood donor. It could not be influenced by a mitogen or by interleukin 2. The study shows that large B lymphocytes with high negative surface charge density are in a later maturation stage than those with lower negative surface charge density.

Sizing of Cells by the Electrical Resistance Pulse Technique

Cells are the constructive elements of all complex biological organisms. It is evident that the size and number of the basic elements define the size of the complete organism or the size of the organs of the organism. Growth of an organism is caused by the growth of the cells, by an increase of the number of cells, or by both. By counting and sizing cells, such fundamental events can be investigated.

A PC-at based video device for flow cytometrically triggered cell imaging in flow.

A personal computer-based system of imaging in flow is described which takes cell images directly from the transducer of a flow cytometer. Imaging is triggered by the pulses of the flow system. The movement of the cells is frozen by ultra-short flashes from pulsed light emitting diodes (LED). The images are taken up by a video camera, transferred to an add-in board of the PC and then stored on disk. A novel method has been developed for capturing the video images which normally arise in asynchronism with the video frame. Images of different cells and particles exposed during flow analysis are shown.