Electrofusion Technique Research Articles

Microwave heating of graphene nanoplatelet polymer composites: Experimental and finite element study

AbstractCompared with contemporary electrofusion techniques that use embedded wires for Joule heating, microwave heating may facilitate the joining of thermoplastic polymer components, promising shortened fusion periods, superior heating uniformity, and reduced energy consumption. This study investigates the fabrication of multifunctional polylactide acid (PLA) composites with strong microwave absorption using graphene nanoplatelets (GNP). GNP/PLA nanocomposites were fabricated using a two‐step scalable manufacturing method, that is, solution blending and hot compression molding. The GNP content of the composites ranged from 0% to 8% by weight. The samples were characterized for dielectric permittivity, heat capacity, and electrical and thermal conductivity. Thermal imaging was used to investigate the efficacy of microwave heating in GNP/PLA nanocomposites as a function of microwave power and filler weight fractions. The microwave heating process in GNP composites was studied using multi‐physics finite element software. The experimental results were compared to numerical model predictions for maximum temperature and microwave energy absorbed. The produced nanocomposites were discovered to have strong microwave absorption characteristics and hence rapid heating, making this type of composite a prospective choice for gasket materials that facilitate fusion bonding for thermoplastic‐based components via localized heating.

Cell electrofusion to improve efficacy and thermotolerance of the entomopathogenic fungus, Beauveria bassiana.

The aim of this study was to optimize the potential of Beauveria bassiana through cell electrofusion technique with emphasis on improving its thermotolerance and virulence against western flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae) (WFT). Nine B. bassiana isolates were tested against western flower thrips, F. occidentalis (Thysanoptera: Thripidae) (WFT) to estimate their pathogenicity and speed of infection under laboratory conditions. The isolates were also exposed to 25, 36, 38 and 40°C, for 24, 48, 72 and 120h. Two isolates with the highest virulence against WFT and highest thermotolerance were selected and paired using a cell electrofusion technique to obtain a single strain that was both highly virulent and thermotolerant. To determine the hybrid conidia among the fusion cells, each selected isolate was loaded with a different nuclei-staining fluorescent dye before the fusion process. Hybridized fungal strains were acquired by a series of sequential dilutions and were tested for thermotolerance and virulence against WFT. Those that demonstrated both characteristics (high thermotolerance and virulence) similar to or greater than the parental isolates were considered hybrids. The results demonstrated that genetic transformation using cell electrofusion can be used to obtain hybrid isolates of fungal pathogens that contain the desired genes from selected isolates. Further DNA-based analysis is required to determine the genetic variation between hybrids and the parental isolates. This is the first study to demonstrate genetic transformation in B. bassiana by use of cell electrofusion and it could be developed to create superior hybrid isolates with various desired properties for integrated pest management and industrial uses in the future.

BREEDING AND DEVELOPMENT OF NOVEL HYBRIDS OF WAXFLOWERS BY PROTOPLAST FUSION

The genus Chamelaucium is a member of Myrtaceae and is endemic to the southwest of Western Australia. Plants from this genus are commonly known as waxflowers. Other related genera in the Chamelaucium tribe include Actinodium, Darwinia, Homoranthus, Pileanthus and Verticordia. Waxflowers are the leading cut flower export from Australia and are grown widely in other countries for the cut-flower industry. At present, most waxflower cultivars are limited to pastel or pale colour shades and to winter-spring flowering periods. To enhance the palette and flowering period of waxflower cultivars we have adopted a strategy of carrying out wide crosses of Chamelaucium species with other members of the Chamelaucium tribe, including Pileanthus and Verticordia. To facilitate these wide crosses we are using the protoplast fusion approach. Purified protoplasts are fused using the technique of electrofusion. To enhance our ability to identify fused populations we used pre-fusion treatments with anti-metabolites, such as iodoacetate, or pre-staining with fluorescent dyes and flow sorting of fused cells post-fusion. Plant regeneration is achieved by somatic embryogenesis of fused cells and fused populations are confirmed by flow cytometry to identify polyploid plants.

PROTOPLAST ELECTRO-FUSION TECHNOLOGY AS A TOOL FOR SOMATIC HYBRIDISATION BETWEEN STRAWBERRIES AND RASPBERRIES

Inter-generic breeding between Fragaria × ananassa and Rubus in order to develop new Phytophthora resistance into Fragaria has been investigated. The study describes the use of the electro-fusion technique allowing to obtain Fragaria × ananassa var. 'Elsanta' (+) Rubus heterocaryons and microcalli. Protoplasts isolation was carried out following the Durieu and Ochatt (2000) procedure. Protoplasts of each species were identified using fluorochromes. Fluorescein diacetate (FDA) and rhodamine B isothiocyanate (RBi) were respectively used for Rubus and Fragaria protoplasts. Under UV light, protoplasts with FDA staining gave a yellow-green fluorescence allowing evaluation of density and viability evaluation while those with RBi gave a red fluorescence. Density and viability were determined for each species. Electro-fusion was achieved using 2 ml cuvettes of an Electro cell Manipulator ECM ® 630 (BTX, California) with electrodes 1 mm apart. Three pulses at 250, 500, 750 or 1000 V·cm -1 were delivered at 10 s intervals (capacitor of 75 μF and variable resistance of 201, 281 or 1540 Ω). The efficiency of protoplast fusion was evaluated under UV light, as the fluorochromes are linked to different parental protoplasts, whereby heterokaryons can be observed and counted through their double fluorescence, green and red. Protoplasts were cultured at 105 cm -3 on a KM medium (Kao and Michayluk, 1975). After one week a dilution was performed with the same medium and, as soon as the majority of cells had regenerated their wall, weekly dilutions (adding weekly 1 ml media per ml of initial protoplast culture) were carried out. Large numbers of heterokaryons have been produced using different Rubus genotypes and different electrical parameters for fusion. Both divisions of heterokaryons and the formation of heterokaryon-derived microcalli were observed.

A method for identification of HIV gp140 binding memory B cells in human blood

Antibodies to HIV are potentially important reagents for basic and clinical studies. Historically, these reagents have been produced by random cloning of heavy and light chains in phage display libraries [Burton, D.R., Barbas, C.F. III, Persson, M.A.A., Koenig, S., Chanock, R.M., and Lerner, R.A., (1991), A large array of human monoclonal antibodies to type 1 immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc. Natl. Acad. Sci. U. S. A. 88, 10134-10137.] and electrofusion techniques [Buchacher, A., Predl, R., Tauer, C., Purtscher, M., Gruber, G., Heider, R., Steindl, F., Trkola, A., Jungbauer, A., and Katinger, H., (1992), Human monoclonal antibodies against gp41 and gp120 as potential agent for passive immunization. Vaccines 92, 191-195]. Here we describe a method to identify and potentially enrich human memory B cells from HIV infected patients that show serum titers of neutralizing antibodies. When biotinylated gp140 is used to stain peripheral blood mononuclear cells it identifies a distinct population of gp140 binding B cells by flow cytometry.

Ultrasonic phased array inspection of electrofusion joints in polyethylene pipes

The electrofusion technique for joining polyethylene pipe is one of the common joining methods used in the water and gas industries for distribution pipes. Common problems in manufacture of these joints are contamination, lack of fusion, lack of penetration and insufficient heat applied to the weld resulting in a sticking or weak bond. The latter flaws can result in a joint that, although apparently fused, does not have the same mechanical properties as a properly prepared joint. The phased array technique has been specifically designed to provide a rapid inspection method for field application and this paper presents an ultrasonic technique for detecting and monitoring flaws in the weld. The results of the extensive laboratory experiments and the field application of the prototype system are presented.

Microfluidic cell fusion under continuous direct current voltage

The authors report a technique which produces cell-to-cell electrofusion using a common direct current power supply on a microfluidic platform. In the authors’ method, the cells were first conjugated based on biotin-streptavidin interaction. The electrofusion was then conducted by flowing the linked cells through a simple microfluidic channel with geometric variation under continuous direct current voltage. This microfluidics-based technique offers processing at the level of single cell pairs with efficiency comparable to that of conventional electrofusion technique based on electrical pulses.

Induction of cytotoxic T lymphocytes against human cancer cell lines using dendritic cell-tumor cell hybrids generated by a newly developed electrofusion technique

Recently, dendritic cells (DCs) and DC-tumor cell hybrids (DC-tumor hybrids) have been used for cancer vaccine therapy in a clinical trial. DC-tumor hybrids combine the potent antigen-presenting capacity of DCs with the ability to present all tumor antigens expressed on tumor cells to T cells. We used DC-tumor hybrids as stimulator cells to induce tumor-specific cytotoxic T lymphocytes (CTLs) in vitro. DC-tumor hybrids were generated from human monocyte-derived DCs and human cancer-cell lines (GT3TKB, lung cancer; GCIY, gastric cancer) by our newly developed electrofusion technique, established and refined with the use of mouse cells. To evaluate the capacity of DC-tumor hybrids generated by our method to induce tumor antigen-specific CTLs, we performed a cytotoxic assay and an interferon-gamma release assay using CD8-dominant effector lymphocytes induced by them. DC-tumor hybrids more effectively induced tumor-specific primary T-cell response than did stimulation with DCs co-cultured with irradiated tumor cells overnight, irradiated tumor cells alone, or a mixture of DCs and irradiated tumor cells. DC-tumor hybrids were generated at a high fusion rate by our electrofusion technique. When CTLs were induced by DC-tumor hybrids in vitro, the high fusion rate did not contribute to the induction of CTLs with increased tumor-specific cytotoxicity. The addition of interleukin-12 to the culture medium did not augment the cytotoxicity of CTLs. Overall, our results suggest that DC-tumor hybrids effectively induce human tumor-specific CTLs and may thus be applicable for clinical trials of adoptive immunotherapy.

Protoplast electrofusion between common wheat (Triticum aestivum L.) and Italian ryegrass (Lolium multiflorum Lam.) and regeneration of mature cybrids

This study describes the development of electrofusion techniques using the ‘donor-recipient’ model for the production of cybrids between common cultivated winter wheat (Triticum aestivum L.) cv. Jinghua No. 1 and a phylogenetically remote, sexually incompatible grass species, Italian ryegrass (Lolium multiflorum Lam.), which belong to two different subtribes: Triticinae and Loliinae. Wheat protoplasts were metabolically inactivated by iodoacetamide before fusion, while protoplasts of Italian ryegrass were X-ray irradiated before protoplast isolation. The suspension cells were directly used to optizmize the inactivation parameters. By exploring the minimum irreversible membrane breakdown strength, the electrofusion parameters were optimized just a few minutes before electrofusion began. A total of 108 green plantlets were obtained, and about half of the green plants uncontrollably necrotized. Among all green plants, 14 were rooted normally and transplanted in growth chamber or field and developed to maturity. All these transplanted plants were male sterile with smaller and off-white anthers. Seeds were obtained by crossing with Jinghua No. 1. Three transplanted regenerants possessed the characteristics of glume facing the rachis, which was the taxonomic characteristic distinguishing the two subtribes of Triticinae and Lolliinae. Although Southern blot hybridization analysis of 33 randomly selected regenerants using a wheat ribosomal DNA probe (pHA71) did not find any differences to wheat, analysis using two mitochondrial probes B342 (cox l), 490 (Pro II) and one chloroplastidic probe pHve H5 revealed that 31 plants were ‘true cybrids’ by showing ryegrass-specific band(s) or new band(s). It also showed that the mitochondria and chloroplasts were not coexistent as the restriction fragment length polymorphism band of Italian ryegrass was not detected by the mitochondrial probes 7 (26s), B342 (cox I), pHJ2-7-1 (cox II), B30 (atp9), and the chloroplast probe pHvc P5. To regenerate the cybrids, the regeneration capacity of the recipient (wheat) was crucial in this study.

81 MOUSE CLONING BY USING A LASER-ASSISTED ZONA OPENING AND ELECTRO-FUSION TECHNIQUE

Mouse cloning can be performed by a direct microinjection of donor nuclei using a conventional or a piezo-actuated technique (Rybouchkin et al. 2002 Reproduction 124, 197–207; Wakayama et al. 1998 Nature 394, 369–374). However, a high percentage of lysed oocytes was observed during the pipette penetration of the cytoplasmic membrane through the zona pellucida. The aim of this experiment was to investigate the possibility of a combination of a laser-assisted zona opening and electro-fusion for mouse cloning. Mature oocytes were obtained from FSH-superovulated B6D2F1 female mice. Enucleation and transfer of donor cell were performed in HEPES-buffered CZB medium. Spindle-chromosome complexes (SCCs) together with first polar body were removed by blunt-end pipette via a small hole in the zona pellucida which was cut by a laser beam. An adult fibroblast cell was introduced into the perivitelline space and fused to the enucleated oocyte by using a single DC pulse of 1.5 kV cm-1, 20 �s, in a fusion medium (Liu and Aoki 2003 Animal Sci. J. 75, 125–129). The fusion rate was checked 30 min later and only the fused oocytes were subjected to activation by 6 h culture in Ca2+-free CZB medium supplemented with 10 mM Sr2+ and 5 �g mL-1 cytochalasin B. The oocytes which presented the pseudo-pronuclei were considered as the activated oocytes and were cultured in CZB medium at 37�C, 5% CO2 in humidified atmosphere. The developmental rate was observed every 24 h for 4 days. The diploid parthenogenetically activated embryos serving as a control were obtained using the same activation protocol but without SCC removal. The percentages of survival after enucleation and after fusion were recorded. The formation of pseudo-pronuclei and the embryos developing to a particular stage were determined by chi-square analysis. The results show that most of the oocytes survived after enucleation (92.5%, 172/186) and the fusion rate was 71.9% (105/146). The formation of pseudo-pronuclei and the cleavage rate of cloned embryos was lower than in the control (87.6% (92/105) vs. 100% (90/90) and 69.6% (64/92) vs. 92.2% (83/90), respectively). The developmental rate to morula–blastocyst stage of cloned embryos was significantly lower than in the control [1.1% (1/92) vs. 44.4% (40/90); P < 0.05]. These results indicate that using laser-assisted zona opening and electro-fusion technique is practical for mouse cloning and provides an alternative method when injection of donor nuclei into the recipient oocytes using a conventional or a piezo-driven method is technically difficult. This study was supported by grants from The National Research Council of Thailand and The Thailand Research Fund (Loyal Golden Julilee Ph.D. program).

Protoplast Fusion in Banana (Musa spp.): Comparison of Chemical (PEG: Polyethylene Glycol) and Electrical Procedure

Optimization of protoplast fusion parameters is a prerequisite for the establishment of somatic fusion technology for banana breeding. In the present investigations, we compared the most frequently used fusion methods: the electrofusion technique and chemical procedure (polyethylene glycol). With regard to frequency of binary fusion, protoplast fusion with the fusogen polyethylene glycol was best. Conversely, electric fusion was found to be better with respect to mitotic activities, somatic embryogenesis and plantlet regeneration rate.

Tumor–dendritic cell fusion as a basis for cancer immunotherapy

Problem: Patients with advanced head and neck cancer are at risk for metastasis - local and regional recurrence. One approach to addressing these issues is immunotherapy. Recent studies have identified the dendritic cell (DC) as a critical component in eliciting a T-cell response against neoplastic cells. One immunotherapy approach is fusion of DCs with irradiated tumor cells by electrofusion. Methods: Fusion cells were created using electrofusion technique. We used 2 allogeneic murine tumor lines (D5 and 4T1) virally transduced to express the antigen B-galactosidase. Using beta-galactosidase as a surrogate tumor marker, we crossed immunized mice with irradiated tumor or fusion cells with subsequent tumor challenge intradermally. Fusion cells were also used in the treatment of three-day established pulmonary metastasis. Results: Cross immunization was achieved with irradiated allogenic tumor cells sharing a common antigen given subcutaneously and intranodally. Successful electrofusion of DC and tumor cells was confirmed using fluorescent activated cell sorting (FACS) and cytospin. Significant responses were demonstrated in immunized mice against tumor challenge and established 3-day pulmonary metastasis with syngeneic-fusion cells. Conclusion: Allogeneic tumors sharing a common tumor antigen can immunize against syngeneic tumor challenge. The use of syngeneic tumor-DC fusion cells demonstrated successful immunization against tumor challenge. Fusion cells also resulted in regression of 3-day established pulmonary metastasis. Significance: Difficulties are encountered in successfully and reliably generating tumor cell lines from individual patients, especially in head and neck cancer patients. Findings over recent years suggest that many tumors of the same histological origin but from different individuals may express common antigens. Therefore, a tumor from a different individual (allogeneic) may be feasible for use as a source of antigens. These preclinical studies provide evidence that an allogeneic tumor-dendritic cell fusion vaccine may be a valid approach for head and neck cancer immunotherapy. Support: American Academy of Otolaryngology-Head and Neck Surgery Resident Research Award

Electrofusion of syngeneic dendritic cells and tumor generates potent therapeutic vaccine

Antigen presentation by dendritic cells (DCs) has the potential to elicit therapeutic immune responses against malignant tumors. One strategy utilizing DC-tumor fusion hybrids as cancer vaccine is particularly attractive because of polyclonal presentation of a diverse array of unaltered tumor antigens. We have recently developed a large-scale electrofusion technique for generating DC-tumor heterokaryons and demonstrated their superb immunogenicity. Here, employing the weakly immunogenic MCA205 sarcoma, a single vaccination with electrofusion hybrids eradicated tumors established in the lung, skin, and brain. Immunotherapy required intra-lymphoid vaccine delivery and co-administration of adjuvants such as OX-40R antibody. Tumor eradication was immunologically specific and involved the participation of both CD4 and CD8 T cells. Consistent with DC’s functionality of MHC-restriction, the use of syngeneic DCs for fusion was an obligatory requirement. Fusion with allogeneic DCs completely lacked therapeutic effects. These findings provide a strong impetus for treating cancer patients with similarly generated DC-tumor hybrids.

Therapeutic immune response induced by electrofusion of dendritic and tumor cells

To elicit a therapeutic antitumor immune response, dendritic cells (DCs) have been employed as a cellular adjuvant. Among various DC-based approaches, fusion of DCs and tumor cells potentially confers not only DC functionality, but also a continuous source of unaltered tumor antigens. We have recently demonstrated successful generation of fusion hybrids by a large-scale electrofusion technique. The immunogenicity and therapeutic potential of fusion hybrids were further analyzed in a model system of a murine melanoma cell line expressing β-galactosidase (β-gal) as a surrogate tumor antigen. A single vaccination with fusion hybrids plus IL-12 induced a therapeutic immune response against 3-day established pulmonary metastases. This immunotherapy was β-gal specific and involved both CD4 and CD8 T cells. In vitro, fusion hybrids stimulated specific IFN-γ secretion from both CD4 and CD8 immune T cells. They also nonspecifically induced IL-10 secretion from CD4 but not CD8 T cells. Compared to other DC loadings, our results demonstrate the superior immunogenicity of fusion. The current technique of electrofusion is adequately developed for clinical use in cancer immunotherapy.

Immunogenicity and Therapeutic Efficacy of Dendritic–Tumor Hybrid Cells Generated by Electrofusion

Dendritic cells (DCs) are potent antigen-presenting cells capable of inducing strong immune responses to weak tumor-associated antigens. Among various DC-based approaches, cancer immunotherapy with DC–tumor fusion hybrids offers advantages of polyclonal stimulation of a diverse array of tumor antigens. However, prevalent fusion methods using chemical fusogens such as polyethylene glycol often result in toxicity and low fusion efficiency. In this article, we describe an electrofusion technique, applicable to processing large numbers of cells with consistent and high fusion efficiency. Generation of fusion hybrids was verified by unequivocal experimental evidence. In animal models, fusion hybrids expressed the mature DC-like phenotype. They stimulated both CD4 and CD8 tumor-specific T cells to secrete interferon-γ in vitro. In immunotherapy, a single vaccination with DC–tumor fusion cells along with interleukin-12 as an adjuvant eradicated tumors established in the skin nd lung. These results provide an impetus for treating cancer patients with similarly generated cells.

Molecular analysis of rheumatoid factor (RF)-negative B cell hybridomas from rheumatoid synovial tissue: evidence for an antigen-induced stimulation with selection of high mutated IgVH and low mutated IgVL/lambda genes.

The mutational pattern of IgVH and IgVL genes from synovial tissue B cell hybridomas (n = 8) of patients (n = 4) with rheumatoid arthritis (RA) was analysed, which had been produced by the electrofusion technique without prior in vitro stimulation. The molecular data were correlated with immunohistopathological data and parameters of local disease activity. The IgVH genes of the B cell hybridomas belonged to the VH3 family (DP42; DP47, n = 2; DP53), the VH1 family (DP75), the VH4 family (DP71) and the VH5 family (DP73); 7/7 IgVH genes showed somatic mutations, the R/S ratio (CDR) was > 3 in 4/7 IgVH genes and the mean R/S ratio of all IgVH genes was 9.3 (CDR) and 1.0 (FR), suggesting an antigen-dependent selection. The IgVL/lambda genes belonged to the Vlambda1 family (DPL2, DPL5, DPL8nf), the Vlambda2 family (DPL11, n = 2) and to the Vlambda6 family (IGLV6S1); 6/6 IgVL genes showed somatic mutations, the R/S ratio (CDR) was > 3 in 3/6 IgVL genes and the mean R/S ratio of all IgVL was 3.0 (CDR) and 2.3 (FR), suggesting an antigen-dependent selection. The synovial tissue exhibited germinal centres in the follicles (3/4), with the unique distribution of Ki-M4+ follicular dendritic cells and Ki-67+ proliferating cells and a dominance of IgA+ plasma cells (3/3). All patients were positive for RF in serum and exhibited severe local symptoms (swelling 4/4; warmth 4/4; effusion 2/4), whereas the hybridomas were negative for RF. Since B cell hybridomas showed hypermutation and affinity selection for IgVH and IgVL/lambda genes and the patients exhibited severe local symptoms with germinal centres in synovial tissue, this study indicates that an antigen-driven process is behind the B cell expansion in the synovial tissue of clinically affected joints. These mutated B hybridomas were negative for RF, thus suggesting that antigens different from RF are also involved in the local B cell expansion and in the chronic synovitis of RA.

ATP-sensitive potassium channels and efaroxan-induced insulin release in the electrofusion-derived BRIN-BD11 beta-cell line.

The properties of ATP-sensitive K+ (K(ATP)) channels were explored in the electrofusion-derived, glucose-responsive, insulin-secreting cell line BRIN-BD11 using patch-clamp techniques. In intact cells, K(ATP) channels were inhibited by glucose, the sulfonylurea tolbutamide, and the imidazoline compounds efaroxan and phentolamine. Each of these agents initiated insulin secretion and potentiated the actions of glucose. K(ATP) channels were blocked by ATP in a concentration-dependent manner and activated by ADP in the presence of ATP. In both intact cells and excised inside-out patches, the K(ATP) channel agonists diazoxide and pinacidil activated channels, and both compounds inhibited insulin secretion evoked by glucose, tolbutamide, and imidazolines. The mechanisms of action of imidazolines were examined in more detail. Pre-exposure of BRIN-BD11 cells to either efaroxan or phentolamine selectively inhibited imidazoline-induced insulin secretion but not the secretory responses of cells to glucose, tolbutamide, or a depolarizing concentration of KCl. These conditions did not result in the loss of depolarization-dependent rises in intracellular Ca2+ ([Ca2+]i), K(ATP) channel operation, or the actions of either ATP or efaroxan on K(ATP) channels. Desensitization of the imidazoline receptor following exposure to high concentrations of efaroxan, however, was found to result in an increase in SUR1 protein expression and, as a consequence, an upregulation of K(ATP) channel density. Our data provide 1) the first characterization of K(ATP) channels in BRIN-BD11 cells, a novel insulin-secreting cell line produced by electrofusion techniques, and 2) a further analysis of the role of imidazolines in the control of insulin release.

Electropermeabilization and electrofusion of human cells modified by anaesthetic agents

Results on the interaction between anaesthetics and human cell membranes are presented in order to show their target behavior including changes in electropermeabilization and resealing. Using electroporation and electrofusion techniques, it is possible to characterize the effects of anaesthetic agents which act as modifiers of cell membrane permeability depending on their types, concentrations and contact times. The field-induced uptake of trypan blue, used as a reporter of membrane electropermeabilization, and electrofusion of human lymphoma cells U 937 and human breast cancer cells MCF 7 were investigated. The cell electropermeabilization decreased with increasing external medium conductivity and osmolarity. The resealing kinetics of both types of cells studied could be fitted by exponentially decaying curves, however the first order rate constant for lymphoma cells was about two orders of magnitude higher. Moreover, the resealing step was slowed down in the presence of anaesthetics. It is suggested that the impact of anaesthetics on cell membranes may be explained by the combined action of different interaction mechanisms, electrical and/or mechanical in nature, as well as by specific bindings to integral membrane molecules, depending on the anaesthetic agent applied. It is concluded that the influence of anaesthetics on in vitro cell electropermeabilization might be used as an additional indicator for clinical purposes.

Flow cytometric detection and quantitation of cell-cell electrofusion products.

AbstractCell-cell electrofusion (CCE) is a process that involves forcing cells into close juxtaposition and then inducing fusion by delivering electric pulses to the cells. CCE has proven to have many practical applications. It has been used for monoclonal antibody (Mab) production (4,5), hybridoma production (1–3), and membrane surface marker transfer (6). Many other applications are described in this volume. In addition, the study of membrane fusion mechanisms has been the focus of some researchers (7–12). Electrofusion techniques seldom result in 100% yields between fusion partners. Therefore, a major aspect of all CCE applications is the ability to detect and quantitate fusion products.KeywordsMicroscopic MethodFusion ProductFusion PartnerHybrid PopulationDual FluorescenceThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Generation of monoclonal progesterone antibodies by electrofusion techniques

The generation of monoclonal antibodies by electrofusion techniques has advanced the efficiency of B-cell immortalisation over ‘PEG’ fusion techniques by greater than 100 fold and also allows for the manipulation of hundreds of cells compared to millions as in the later technique. Most researchers in this field at present have equipment which is highly specialised and machined ‘in-house’ or expensive commercial electrofusion apparatus which precludes small laboratories with limited access to either machine shops or research facilities from using this technique. We present here data showing that it is possible to make a small switching device and couple this to readily available commercial electroporation instruments making an efficient electrofusion device that a competent electronics technician could accomplish readily.