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Chris R Bleackley - One of the best experts on this subject based on the ideXlab platform.
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peritoneal exudate lymphocyte and mixed lymphocyte culture Hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin
European Journal of Immunology, 1992Co-Authors: Cheryl D Helgason, John A Prendergast, Gideon Berke, Chris R BleackleyAbstract:We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) Hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five Hybridoma clones tested. With the exception of low level CCP2 expression in the MLC Hybridoma MD45 following antigen stimulation, all of the Hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.
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peritoneal exudate lymphocyte and mixed lymphocyte culture Hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin
European Journal of Immunology, 1992Co-Authors: Cheryl D Helgason, John A Prendergast, Gideon Berke, Chris R BleackleyAbstract:: We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) Hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five Hybridoma clones tested. With the exception of low level CCP2 expression in the MLC Hybridoma MD45 following antigen stimulation, all of the Hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.
Cheryl D Helgason - One of the best experts on this subject based on the ideXlab platform.
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peritoneal exudate lymphocyte and mixed lymphocyte culture Hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin
European Journal of Immunology, 1992Co-Authors: Cheryl D Helgason, John A Prendergast, Gideon Berke, Chris R BleackleyAbstract:We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) Hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five Hybridoma clones tested. With the exception of low level CCP2 expression in the MLC Hybridoma MD45 following antigen stimulation, all of the Hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.
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peritoneal exudate lymphocyte and mixed lymphocyte culture Hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin
European Journal of Immunology, 1992Co-Authors: Cheryl D Helgason, John A Prendergast, Gideon Berke, Chris R BleackleyAbstract:: We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) Hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five Hybridoma clones tested. With the exception of low level CCP2 expression in the MLC Hybridoma MD45 following antigen stimulation, all of the Hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.
Gideon Berke - One of the best experts on this subject based on the ideXlab platform.
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peritoneal exudate lymphocyte and mixed lymphocyte culture Hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin
European Journal of Immunology, 1992Co-Authors: Cheryl D Helgason, John A Prendergast, Gideon Berke, Chris R BleackleyAbstract:We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) Hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five Hybridoma clones tested. With the exception of low level CCP2 expression in the MLC Hybridoma MD45 following antigen stimulation, all of the Hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.
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peritoneal exudate lymphocyte and mixed lymphocyte culture Hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin
European Journal of Immunology, 1992Co-Authors: Cheryl D Helgason, John A Prendergast, Gideon Berke, Chris R BleackleyAbstract:: We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) Hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five Hybridoma clones tested. With the exception of low level CCP2 expression in the MLC Hybridoma MD45 following antigen stimulation, all of the Hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.
John A Prendergast - One of the best experts on this subject based on the ideXlab platform.
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peritoneal exudate lymphocyte and mixed lymphocyte culture Hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin
European Journal of Immunology, 1992Co-Authors: Cheryl D Helgason, John A Prendergast, Gideon Berke, Chris R BleackleyAbstract:We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) Hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five Hybridoma clones tested. With the exception of low level CCP2 expression in the MLC Hybridoma MD45 following antigen stimulation, all of the Hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.
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peritoneal exudate lymphocyte and mixed lymphocyte culture Hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin
European Journal of Immunology, 1992Co-Authors: Cheryl D Helgason, John A Prendergast, Gideon Berke, Chris R BleackleyAbstract:: We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) Hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five Hybridoma clones tested. With the exception of low level CCP2 expression in the MLC Hybridoma MD45 following antigen stimulation, all of the Hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.
Edward A. Greenfield - One of the best experts on this subject based on the ideXlab platform.
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Single-Cell Cloning of Hybridoma Cells by Limiting Dilution.
Cold Spring Harbor protocols, 2019Co-Authors: Edward A. GreenfieldAbstract:Isolating a stable clone of Hybridoma cells that all secrete the correct antibody is the most time-consuming step in the production of Hybridomas. Single-cell cloning ensures that cells that produce the antibody of interest are truly monoclonal and that the secretion of this antibody can be stably maintained. The original positive well will often contain more than one clone of Hybridoma cells, and many hybrid cells have an unstable assortment of chromosomes. Both of these problems may lead to the desired cells being outgrown by cells that are not producing the antibody of interest. Cloning Hybridoma cells by limiting dilution is the easiest of the single-cell-cloning techniques. Two approaches are presented here, one rapid technique for generating cultures that are close to being single-cell-cloned and one for single-cell cloning directly. Even though every attempt is made to ensure that the cells are in a single-cell suspension before plating, there is no way to guarantee that the colonies do not arise from two cells that were stuck together. Therefore, limiting dilution cloning should be performed at least twice to generate a clonal population.
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preparing feeder cell cultures to support Hybridoma growth
CSH Protocols, 2019Co-Authors: Edward A. GreenfieldAbstract:Feeder layer plates are prepared with cells that provide the appropriate growth factors to support the growth of Hybridoma cells until they can expand in number and provide their own. Good feeder cells should have properties that allow them to be selected against during the future growth of the Hybridomas. Peritoneal macrophages, myeloma cells, splenocytes, and MRC-5 cells (a human lung fibroblast line) are the most common feeder layer cells used in Hybridoma fusions. Methods for their preparation are described here.
