The Experts below are selected from a list of 1398 Experts worldwide ranked by ideXlab platform
David M. Lewinsohn - One of the best experts on this subject based on the ideXlab platform.
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Generation of MR1-Restricted T Cell Clones by Limiting Dilution Cloning of MR1 Tetramer+ Cells.
Methods in molecular biology (Clifton N.J.), 2019Co-Authors: Meghan E. Cansler, Megan D. Null, Erin W. Meermeier, Gwendolyn Swarbrick, David M. Lewinsohn, Melanie J. HarriffAbstract:Tetramers are a powerful tool for identification of T cell subsets that are restricted by specific antigen presenting molecules and their cognate antigens. The generation of T cell clones from specific T cell subsets allows for further investigation of the phenotype and function of these cells. Here, we describe a method for sorting and Cloning of MR1-restricted T cells using the MR1/5-OP-RU tetramer. This protocol can be easily modified to enrich for expansion of specific or unique subsets of MR1-restricted T cell clones from any tissue to further characterize the phenotype and function of those cells.
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Human Dendritic Cells Presenting Adenovirally Expressed Antigen Elicit Mycobacterium tuberculosis–Specific CD8+ T Cells
American journal of respiratory and critical care medicine, 2002Co-Authors: Deborah A. Lewinsohn, Rebecca A. Lines, David M. LewinsohnAbstract:Previous studies in murine and human models have suggested an important role for CD8+ T cells in host defense to Mycobacterium tuberculosis (Mtb). Consequently, a successful tuberculosis vaccine may require the elicitation of sustained CD4+ and CD8+ T cell responses. We tested the hypothesis that the potent CD4+ T cell antigen Mtb39 is also a CD8+ T cell antigen. A recombinant adenovirus–expressing Mtb39 (adenoMtb39) was used to infect monocyte-derived dendritic cells. Using interferon-γ enzyme-linked immunospot, Mtb39-specific CD8+ T lymphocytes were detected in three healthy individuals with latent tuberculosis infection who also had strong anti–Mtb39-specific CD4+ T cell responses. An Mtb39-specific CD8+ T cell line was generated using Mtb39-expressing dendritic cells. Mtb39-specific T cell clones were obtained by limiting Dilution Cloning. All seven T cell clones obtained were HLA-B44 restricted. Using a panel of synthetic overlapping peptides representative of Mtb39, the peptide epitope was identifie...
Marcus Lee - One of the best experts on this subject based on the ideXlab platform.
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Defining multiplicity of vector uptake in transfected Plasmodium parasites
Scientific Reports, 2020Co-Authors: Manuela Carrasquilla, Theo Sanderson, Ruddy Montandon, Alena Pance, Sophie Adjalley, Alejandro Marin-menendez, Rachael Coyle, Julian Rayner, Marcus LeeAbstract:The recurrent emergence of drug resistance in Plasmodium falciparum increases the urgency to genetically validate drug resistance mechanisms and identify new targets. Reverse genetics have facilitated genome-scale knockout screens in Plasmodium berghei and Toxoplasma gondii, in which pooled transfections of multiple vectors were critical to increasing scale and throughput. These approaches have not yet been implemented in human malaria species such as P. falciparum and P. knowlesi, in part because the extent to which pooled transfections can be performed in these species remains to be evaluated. Here we use next-generation sequencing to quantitate uptake of a pool of 94 barcoded vectors. The distribution of vector acquisition allowed us to estimate the number of barcodes and DNA molecules taken up by the parasite population. Dilution Cloning of P. falciparum transfectants showed that individual clones possess as many as seven episomal barcodes, revealing that an intake of multiple vectors is a frequent event despite the inefficient transfection efficiency. Transfection of three spectrally-distinct fluorescent reporters allowed us to evaluate different transfection methods and revealed that schizont-stage transfection limited the tendency for parasites to take up multiple vectors. In contrast to P. falciparum, we observed that the higher transfection efficiency of P. knowlesi resulted in near complete representation of the library. These findings have important implications for how reverse genetics can be scaled in culturable Plasmodium species.
Melanie J. Harriff - One of the best experts on this subject based on the ideXlab platform.
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Generation of MR1-Restricted T Cell Clones by Limiting Dilution Cloning of MR1 Tetramer+ Cells.
Methods in molecular biology (Clifton N.J.), 2019Co-Authors: Meghan E. Cansler, Megan D. Null, Erin W. Meermeier, Gwendolyn Swarbrick, David M. Lewinsohn, Melanie J. HarriffAbstract:Tetramers are a powerful tool for identification of T cell subsets that are restricted by specific antigen presenting molecules and their cognate antigens. The generation of T cell clones from specific T cell subsets allows for further investigation of the phenotype and function of these cells. Here, we describe a method for sorting and Cloning of MR1-restricted T cells using the MR1/5-OP-RU tetramer. This protocol can be easily modified to enrich for expansion of specific or unique subsets of MR1-restricted T cell clones from any tissue to further characterize the phenotype and function of those cells.
Mohamed Al-rubeai - One of the best experts on this subject based on the ideXlab platform.
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The selection of high-producing cell lines using flow cytometry and cell sorting
Expert opinion on biological therapy, 2004Co-Authors: Silvia Carroll, Mohamed Al-rubeaiAbstract:The selection of high-producing cell lines is usually time-consuming and labour-intensive. Following transfection, high-producing cells are selected using limiting Dilution Cloning to prevent non- and low-producing cells from outgrowing high-producing cells, a process that normally takes > 3 months. During this time, the cells have to be screened occasionally to ensure stability of the selected clone. Several new methods for selecting and screening cells using flow cytometry and cell sorting have recently been developed; these include gel microdrop technology, which encapsulates the cells in gelatine beads, and matrix-based secretion assays. This paper reviews these techniques for selecting high-producing cell lines and isolating rare cells.
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Chromosome instability in Spodoptera frugiperda Sf-9 cell line.
Biotechnology progress, 2002Co-Authors: R. F. Jarman‐smith, S. J. Armstrong, C. J. Mannix, Mohamed Al-rubeaiAbstract:Homogeneous cell lines are essential in industry and research if reliable and reproducible data are to be obtained. The Spodoptera frugiperda (Sf-9) cell line routinely used for the production of recombinant proteins was found to be heterogeneous, containing a mixture of diploid and tetraploid cells. Using Dilution-Cloning techniques, diploid and tetraploid subpopulations were isolated from a Sf-9 parental cell line, and their cytogenetic state was monitored using Vinblastine to arrest cells in mitosis. Flow cytometry was used to obtain a snapshot of the predominant subpopulations present to verify the karyological results. The rate at which clonal populations digress into the heterogeneous state was found to be more rapid for the diploid subpopulation, with the emergence of tetraploid cells after only 11 passages, than for the tetraploid subpopulation, where diploid clones appeared after 18 passages. The chromosomes in both diploid and tetraploid subpopulations as well as the parental cell line were found to spontaneously fragment during growth and expansion processes, giving rise to variable chromosome numbers. DNA analysis of cell lines obtained from laboratories worldwide have shown that the Sf-9 cell line used for the production of many recombinant proteins is cytologically unstable, leading to varying degrees of polyploidal state depending on its culture history and supplier.
Manuela Carrasquilla - One of the best experts on this subject based on the ideXlab platform.
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Defining multiplicity of vector uptake in transfected Plasmodium parasites
Scientific Reports, 2020Co-Authors: Manuela Carrasquilla, Theo Sanderson, Ruddy Montandon, Alena Pance, Sophie Adjalley, Alejandro Marin-menendez, Rachael Coyle, Julian Rayner, Marcus LeeAbstract:The recurrent emergence of drug resistance in Plasmodium falciparum increases the urgency to genetically validate drug resistance mechanisms and identify new targets. Reverse genetics have facilitated genome-scale knockout screens in Plasmodium berghei and Toxoplasma gondii, in which pooled transfections of multiple vectors were critical to increasing scale and throughput. These approaches have not yet been implemented in human malaria species such as P. falciparum and P. knowlesi, in part because the extent to which pooled transfections can be performed in these species remains to be evaluated. Here we use next-generation sequencing to quantitate uptake of a pool of 94 barcoded vectors. The distribution of vector acquisition allowed us to estimate the number of barcodes and DNA molecules taken up by the parasite population. Dilution Cloning of P. falciparum transfectants showed that individual clones possess as many as seven episomal barcodes, revealing that an intake of multiple vectors is a frequent event despite the inefficient transfection efficiency. Transfection of three spectrally-distinct fluorescent reporters allowed us to evaluate different transfection methods and revealed that schizont-stage transfection limited the tendency for parasites to take up multiple vectors. In contrast to P. falciparum, we observed that the higher transfection efficiency of P. knowlesi resulted in near complete representation of the library. These findings have important implications for how reverse genetics can be scaled in culturable Plasmodium species.
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Quantitation of vector uptake reveals non-Poissonian transfection dynamics in Plasmodium falciparum
2019Co-Authors: Manuela Carrasquilla, Theo Sanderson, Ruddy Montandon, Julian C. Rayner, Alena Pance, Marcus C. S. LeeAbstract:Abstract The recurrent emergence of drug resistance in Plasmodium falciparum increases the urgency to genetically validate drug resistance mechanisms and identify new targets. Reverse genetics have facilitated genome-scale knockout screens in Plasmodium berghei and Toxoplasma gondii, in which pooled transfections of multiple vectors were critical to increasing scale and throughput. These approaches have not yet been implemented in P. falciparum, mainly because the extent to which pooled transfections can be performed in this species still remains unknown. Here we use next-generation sequencing to quantitate uptake of a pool of 94 barcoded vectors. The distribution of vectors in different transfections allowed us to estimate the number of barcodes and DNA molecules taken up by the parasite population. Dilution Cloning showed that single transfected parasites routinely carry as many as seven episomal barcodes, revealing an intake of multiple vectors in a highly non-uniform fashion. Transfection of non-overlapping fluorescent proteins, which allowed us to follow the dynamics of the process, confirmed the tendency for parasites to take up multiple vectors from the early stages of transfection. This finding has important implications for how reverse genetics can be scaled in P. falciparum.