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FCCH100156 | FlowCellect™ Human T Cell Caspase 9 Kit

FCCH100156
100 Tests / Kit  
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      Replacement Information
      Description
      Catalogue NumberFCCH100156
      Trade Name
      • FlowCellect
      DescriptionFlowCellect™ Human T Cell Caspase 9 Kit
      Overview Millipore’s FlowCellect™ Human T Cell Caspase 9 Kit provides a simplified and rapid method to assess apoptosis based on Caspase 9 activity in sub-populations of T cells. The CD4-PE/CD3-PECy5/CD8-APC cocktail consists of three anti-human antibodies and includes anti-human CD3, CD4 and CD8 antibodies which allow for T cell detection and identification. The CD3 antibody Anti-CD3 (Clone UCHT1), reacts with the ε-chain of the CD3 part of the TCR/CD3 complex. CD3 is a pan-T marker expressed by normal and neoplastic T cells and uniquely allows the identification of all T cell lymphocytes. The Anti-Human CD4 Antibody (Clone MT310) allows the identification of human helper/inducer CD4+ T cell (HLA Class II reactive) and recognizes a 55 kDa glycoprotein on the surface of CD4 T helper cells. Monocytes also express CD4 but at lower density, and have no co-expression of CD3 and hence can be distinguished away from CD4 T Cells when using this kit. The Anti-human CD8 antibody (Clone DK-25) allows the identification of CD8, a 68 kDa, disulfide linked transmembrane glycoprotein expressed by class I major histocompatibility complex restricted, mature suppressor/cytotoxic T cells, the great majority of cortical thymocytes and approximately 30% of medullary thymocytes. In addition a proportion of γδ T cells and NK cells express CD8. Inclusion of the Anti-CD3 antibody allows for the unique identification of the CD8 cytotoxic T Cells when using the kit.

      Caspase 9 reagent is a fluorescent inhibitor of caspases and is membrane permeable. The Caspase 9 reagent (FAM-LEHD-FMK) consists of 3 parts; a caspase-specific amino acid inhibitor containing a recognition sequence for Caspase 9 (LETD), a fluoromethylketone moiety (FMK) that forms a covalent linkage with the active enzyme; and and a carboxyfluorescein (FAM) reporter. The Caspase 9 reagent binds to caspases which are activated during apoptosis with resulting fluorescent signal proportional to the number of active caspases in cell (13, 14). Due to the presence of FAM reporter , caspase activity is observed in the green channel of flow cytometers. Thus, in apoptotic cells, due to the activation of Caspase 9 during the apoptotic process, there is increased green fluorescence observed due to the increased caspase activity in these cells.

      The FlowCellect Human T Cell Caspase 9 kit can thus distinguish multiple populations (1) CD4 T Helper cells (2) % of CD4 T Helper cells which show Caspase 9 activity (3) CD8 cytotoxic T Cells and (4) % of CD8 cells which show Caspase 9 activity. The kit thus provides a complete picture of T cell apoptotic status and its response for inducer treatment conditions or diseases. The entire assay can be performed in <90 min with a single incubation step when using PBMC’s.
      Alternate Names
      • Flow Cellect
      Background InformationApoptosis plays a critical role in development, regulation and functioning of the immune system and is also a contributor to the pathology of many immune diseases and disorders. Apoptosis provides the mechanism for deletion of autoreactive T cells in the thymus, low responsive B cells in the germinal center, and of target cells attacked by cytotoxic T lymphocytes and natural killer cells. The process of apoptosis is accompanied by multiple morphological, biochemical and physiological changes in response to specific induction signals. Among these are externalization of phosphatidylserine to the cell surface, cleavage and degradation of specific cellular proteins by activated enzymes such as caspases, compaction and fragmentation of nuclear chromatin, and loss of membrane integrity (in late stages).

      Caspases are a family of cysteine proteases that play essential roles in apoptosis, necrosis, and inflammation. Caspases are typically expressed as inactive precursors which get activated during apoptosis and cleave substrates after specific aspartate residues. Caspase 9 is an initiator caspase which is critical to the intrinsic pathway of apoptosis. In the intrinsic apoptotic pathway, cellular insults lead to mitochondrial alteration and cytochrome c release.
      In the presence of cytochrome c and dATP or ATP, the adaptor protein Apaf-1 assembles into an oligomeric structure known as the apoptosome, which recruits
      and activates caspase-9. Caspase 9 then activate effector caspases such as caspase-3 and -7, which cleave a wide range of substrates to execute apoptosis.
      References
      Product Information
      Components
      • 1. CD4-PE/CD3-PECy5/CD8-APC (Part No.4700-1420). One vial containing 1000 µL of Antibody Cocktail.
      • 2. Caspase 9 FAM : (Part No. 4100-1380). One vial containing 100 tests worth of lyophilized material
      • 3. 1X Assay Buffer BA: (Part No. 4700-1425) One vial containing 10 mL buffer.
      • 4. 10X Apoptosis Wash Buffer (Part No. 4200-0162) One bottle containing 10 mL buffer.
      • 5. Anhydrous DMSO (Part No. 4300-0160) One vial containing 0.5mL.
      Detection methodFluorescent
      Applications
      ApplicationThis FlowCellect Human T Cell Caspase 9 Kit for Flow Cytometry provides a simplified & rapid method to assess apoptosis based on Caspase 9 activity in sub-populations of T cells.
      Key Applications
      • Flow Cytometry
      Biological Information
      CloneUCHT1; MT310; DK-25
      HostMouse
      Species Reactivity
      • Human
      Antibody TypeMonoclonal Antibody
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Quality AssuranceLot to lot in-process testing on all kit components
      Usage Statement
      • Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
      Storage and Shipping Information
      Storage Conditions4-8°C for antibodies and buffers
      Packaging Information
      Material Size100 Tests / Kit
      Transport Information
      Supplemental Information
      Specifications

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