Dennie Magcase

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Intracellular Staining with MHC Tetramers

Posted by Dennie Magcase on May 27, 2020 1:00:00 PM

T-cells recognize peptides/MHC complexes (pMHC) through the T-cell receptor (TCR). This is the first step for the initiation and shaping of protective immunity against viruses and tumor antigens. Fluorescently labelled pMHC tetramers have dramatically transformed the detection of antigen specific T-cells. MHC tetramer staining of antigen specific T cell clonotypes is detected by flow cytometry to probe T-cell responses1 and to further characterize antigen-specific T cells, for example, to study the surface markers as well as intracellular proteins. The measurement and analysis of effector function of antigen-reactivated T cells is now possible by MHC tetramer and flow cytometry-based intracellular cytokine staining (ICS). This method allows concurrent phenotypic characterization and cytokine detection within single cells2.

The production of cytokines plays an important role in immune response.  Cytokines are involved in many pathways, including the induction of many anti-viral proteins by IFNγ, the induction of T cell proliferation by IL-2, and the inhibition of viral gene expression and replication by TNFα3.  Cytokines are not preformed factors but are produced and secreted in response to cellular activation.

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Topics: Tetramer, cytokine

The problem with Ultracentrifugation for exosome isolation

Posted by Dennie Magcase on Nov 2, 2017 12:00:00 PM

Ultracentrifugation is regarded as the gold standard method for exosome isolation.  The ultracentrifugation process is time consuming and may introduce protein and nucleic acid contamination.  Pre-clearing samples by one or more low speed centrifugation steps will deplete the cells, platelets, and large apoptotic bodies.    Larger extracellular vesicles can be pelleted at forces between 10000-20000 x g.  Smaller extracellular vesicles can be pelleted at higher speeds, ranging from 100000-200000 x g. Depending on the protocol, it can take up to 24 hours to isolate exosomes by ultracentrifugation.  It is impossible to achieve absolute separation due to potential contamination of proteins and nucleic acids.

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Topics: Exosomes, ultracentrifugation

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