Exposure System for Production of Uniform Magnetic Fields

An expression was derived for the magnetic field between Helmholtz coils, and used to determine the intercoil region within which the field's spatial uniformity is below an arbitrary value. Coils were designed to produce a magnetic field uniform to within 5% in a specified volume, and the results of the calculations were verified by direct measurement.     

System for Studying Uninfected and Virus-Infected Cell Cultures in Hyperbaric Chambers

Equipment and techniques previously used to investigate the effect of hyperbaric gases on bacteria were modified to permit comparable investigations with uninfected and virus-infected tissue cell cultures. This report describes the modified equipment and related methodology. Use of the system is illustrated with findings on the effect of oxygen-helium mixtures at 68 atm on cell physiology and virus growth in two cell types. Our results suggested that, under those experimental conditions, several synthetic processes in chick fibroblast monolayers are inhibited but that Sindbis virus growth in the cells is increased. Growth of Japanese encephalitis virus in porcine kidney cells was found to be unaffected by oxygen-helium gas at partial pressures of oxygen between 0 and 700 mm Hg, but morphological alterations in the cells occurred at low and high pO(2) levels.     

Nicotine Tolerance in Chromaffin Cell Cultures: Acute and Chronic Exposure to Smoking-Related Nicotine Doses

Abstract: Nicotine tolerance and dependence are key aspects of tobacco addiction; however, the cellular mechanisms underlying these phenomena are poorly understood. Adrenal chromaffin cells release catecholamines upon exposure to nicotine and with repeated exposure this response exhibits nicotine tolerance. Using bovine adrenal chromaffin cells in culture, we have demonstrated acute and chronic nicotine tolerance at doses relevant to that in the blood and tissues of smokers (10–⁷M to 10–⁶M). Chromaffin cells are preexposed to low doses of nicotine for time periods ranging from 10 min to 7 days and then subsequently challenged with a maximally stimulating dose of nicotine (10–⁵M) for 10 min, all at 37°C. Preexposure to nicotine results in a depression of ⁴⁵Ca uptake and catecholamine release upon subsequent nicotine challenge. Acute tolerance or desensitization of nicotinestimulated catecholamine release begins to occur in minutes after preexposure to 10–⁶M nicotine at 37°C. The depression of catecholamine release upon preexposure to nicotine is both dose and temperature dependent and is not seen with potassium-evoked release. Chronic exposure to 10–⁷M nicotine for 3 days led to a depression of the secretory response to ～70% of control responses. There was a trend toward recovery of full response by days 5 and 7 of 10–⁷M nicotine preexposure. Nearly complete depression of the nicotine-evoked release occurs within the first day of exposure to 10–⁶M nicotine and persists for at least a week of nicotine exposure at 37°C. Nicotine tolerance in the catecholamine release response was reversible after nicotine washout. Cross-tolerance between nicotine, acetylcholine, and dimethylphenylpiperazinium was observed after 5 days’exposure to one agonist and subsequent challenge with a different agonist. Acute tolerance is likely to be related to nicotinic receptor desensitization. The mechanisms of chronic tolerance and potential adaptive cellular changes remain to be determined.     

High-Yield Method for Dispersing Simian Kidneys for Cell Cultures

A technique for dispersion of animal tissue cells is described. The proposed technique is based on the concomitant use of trypsin and disodium ethylenediamine tetraacetate (EDTA). The use of the two dispersing agents (trypsin and disodium EDTA) markedly enhances cell yield as compared with the standard cell dispersion methods. Moreover, significant reduction in the amount of time required for complete tissue dispersal, presence of a very low number of nonviable cells, less cell clumping, and more uniform monolayer formation upon cultivation compare favorably with the results usually obtained with the standard trypsinization technique.     

Two Modes of Cell Death Caused by Exposure to Nanosecond Pulsed Electric Field

High-amplitude electric pulses of nanosecond duration, also known as nanosecond pulsed electric field (nsPEF), are a novel modality with promising applications for cell stimulation and tissue ablation. However, key mechanisms responsible for the cytotoxicity of nsPEF have not been established. We show that the principal cause of cell death induced by 60- or 300-ns pulses in U937 cells is the loss of the plasma membrane integrity (“nanoelectroporation”), leading to water uptake, cell swelling, and eventual membrane rupture. Most of this early necrotic death occurs within 1–2 hr after nsPEF exposure. The uptake of water is driven by the presence of pore-impermeable solutes inside the cell, and can be counterbalanced by the presence of a pore-impermeable solute such as sucrose in the medium. Sucrose blocks swelling and prevents the early necrotic death; however the long-term cell survival (24 and 48 hr) does not significantly change. Cells protected with sucrose demonstrate higher incidence of the delayed death (6–24 hr post nsPEF). These cells are more often positive for the uptake of an early apoptotic marker dye YO-PRO-1 while remaining impermeable to propidium iodide. Instead of swelling, these cells often develop apoptotic fragmentation of the cytoplasm. Caspase 3/7 activity increases already in 1 hr after nsPEF and poly-ADP ribose polymerase (PARP) cleavage is detected in 2 hr. Staurosporin-treated positive control cells develop these apoptotic signs only in 3 and 4 hr, respectively. We conclude that nsPEF exposure triggers both necrotic and apoptotic pathways. The early necrotic death prevails under standard cell culture conditions, but cells rescued from the necrosis nonetheless die later on by apoptosis. The balance between the two modes of cell death can be controlled by enabling or blocking cell swelling.     

Shock Wave Application to Cell Cultures

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to target cells or tissue without any subsequent damage. Therefore, in vitro experiments are of increasing interest. Various methods of applying shock waves onto cell cultures have been described. In general, all existing models focus on how to best apply shock waves onto cells.However, this question remains: What happens to the waves after passing the cell culture? The difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected! We therefore developed a model that mainly consists of a Plexiglas built container that allows the waves to propagate in water after passing the cell culture. This avoids cavitation effects as well as reflection of the waves that would otherwise disturb upcoming ones. With this model we are able to mimic in vivo conditions and thereby gain more and more knowledge about how the physical stimulus of shock waves gets translated into a biological cell signal (“mechanotransduction").     

Intensifier for Bodian Staining of Tissue Sections and Cell Cultures

Intensification of the standard Bodian silver stain with a poststaining photographic enhancer produces high resolution of fine cell processes such as axonal growth cones. This technique can be used on tissue sections and is especially useful for visualizing individual cells fixed in tissue cultures.     

复频声场均匀性的研究

目的：从理论上分析复频声场中不同频率及相位的取值对声场均匀性的影响。方法：给出了平均声压与声压均方差表达式;绘制了不同频率的情况下,平均声压与声压均方差随相位的变化,并对各图进行了具体分析。结果：通过对各参数分析可知,频率及相位的取值不同会影响声波的总能量与均匀性。结论：选取合适的参数可以实现声场均匀性。     

Development of Eimeria auburnensis in Cell Cultures

SYNOPSIS. Monolayer established cell line cultures of bovine kidney (Madin-Darby) and human intestine (Intestine 407), as well as embryonic bovine tracheal and embryonic spleen cell line cultures were inoculated with E. auburnensis sporozoites and observed for a maximum of 22 days. Mature 1st generation schizonts developed in the kidney, tracheal and spleen cells. In the intestine cells, trophozoites were seen in 3 of 4 experiments, but schizonts were not found. Sporozoites penetrated cells, beginning within a few minutes after inoculation. Penetration was usually accomplished within 10 seconds, and the body of the sporozoite underwent a slight constriction as it passed thru the host cell membrane. Some sporozoites left cells. Numerous intracellular sporozoites were observed in kidney, tracheal and spleen cultures. Crescent bodies were seen in the parasitophorous vacuole as early as 1 day after inoculation. At this time, the nuclei of most intracellular sporozoites had changed from vesicular to compact. Beginning 4 days after inoculation, enlarged sporozoites and parasites having a sporozoite shape, but with 2-5 nuclei, were frequently seen. These enlarged sporozoites and sporozoite-shaped schizonts evidently transformed into trophozoites and spheroidal schizonts by means of lateral outpocketings. Few trophozoites were seen. More immature schizonts developed in kidney cells than in the other cell types. The numbers of mature schizonts observed in kidney and tracheal cells were similar, but development occurred less consistently in the latter. Few immature and mature schizonts developed in spleen cells. Mature schizonts, first seen 9 days after inoculation, were considerably smaller than those reported from calves. Some motile merozoites were seen; evidently no development beyond these occurred. The nucleus and nucleolus of host cells were enlarged; this enlargement was not as pronounced as in infections in calves. Multiple host cell nuclei were frequently observed. Degenerative changes in the cultured cells and in the parasites usually occurred, beginning 9-17 days after inoculation; these were more pronounced in the spleen cells than in the others.     

Electromagnetic Field Exposure from Pervasive Computing

With the number of electromagnetic field sources in human environments increasing, it appears appropriate to discuss the future levels of radio frequency (RF) exposure. However, general quantitative assessment is impeded by a fundamental lack of current data. Nevertheless, major technological trends and innovation paths are presented and evaluated with respect to their effect on exposure levels. The most relevant wireless standards and their RF characteristics are briefly introduced. The factors influencing RF exposure are treated in a systematic fashion. A simple exposure-oriented classification of present and future RF sources is introduced to facilitate a structured discussion. The most relevant levers for limiting future exposure levels are presented and some general conclusions are drawn.     

An Embedding Method for Monolayer Cell Cultures for Light and Electron Microscopy

Plastic containers are widely used for monolayer cell culture. In situ embedding, the obvious method of choice for subsequent ultrastructural study, has been achieved by Brinkley et al. (1967) using Epon as a final embedding medium and water soluble acrylates as intermediates. Although the results are satisfactory, the method has two drawbacks: firstly, the water soluble acrylates are difficult to get, and secondly, removal of the plastic container is possible only with blocks already cut off for electron microscopy.     

Callus and Cell Suspension Cultures of Carnation

Callus cultures of carnation, Dianthus caryophyllus L. ev. G. J. Sim, were grown on a synthetic medium of half strength Murashige and Skoog salts, 3 % sucrose, 100 mg/l of myo-inositol, 0.5 mg/l each of thiamin, HCl, pyridoxin, HCl and nicotinic acid and 10 g/l agar. Optimal concentrations of growth regulators were observed to be 3 × 10^?6M indoleacetic acid (JAA) combined with 3 × 10^?6M benzylaminopurin (BAP) or 10^?6M 2,4-dichlorophenoxy acetic acid (2,4-D) alone. IAA + BAP caused a 100 fold increase in fresh weight over 4 weeks at 25°C. Addition of casein hydrolysate increased growth further. Cell suspension cultures worked best in media containing 2,4-D in which they had a doubling time of about 2 days. Filtered suspensions were successfully plated on agar in petri dishes, but division was never observed in single cells. The cultures initiated roots at higher concentrations of IAA or NAA, but all attempts to induce formation of shoots or em-bryoids gave negative results.     

Effect of Coronatine on Plant Cell Cultures

The effect of the phytotoxin coronatine, formed by pathovars of Pseudomonas syringae, on the plant cell cultures of Lycopersicon peruvianum, Lycopersicon esculentum, Chenopodium album and Solanum tuberosum was investigated. The parameters studied were growth, triphenyltetrazolium chloride activity (test for reductases), cell protein content and activities of polyphenol oxidase and peroxidase, both within the cells and in the culture liquid. The cultures of Lycopersicon peruvianum and Lycopersicon esculentum cultures were more sensitive than Chenopodium album and Solanum tuberosum. The more resistant cultures possess a high level of extracellular polyphenol oxidase.     

植物细胞培养生产重组蛋白

用植物细胞培养生产重组蛋白,集合了微生物发酵的快速性、动物细胞培养产物的多样性和完整植株培养系统的安全性,近年来引起了广泛的关注。虽然还未有用植物细胞培养来进行重组蛋白的商业生产,但是它的生产原则较规范,下游处理过程较简单,具有潜在商业生产的可行性。     

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal¹. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal^2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture^4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.Open in a separate windowClick here to view.^{(105M, flv)}     

Plaque Assay for Polyoma Virus on Primary Mouse Kidney Cell Cultures

A plaque assay for polyoma virus using primary baby mouse kidney cells is reported.