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      Z-LEHD-FMK, Caspase-9 Inhibitor
      Z-LEHD-FMK, Caspase-9 Inhibitor
      Flow cytometric analysis of apoptosis in Jurkat cells.  Jurkat cells (Human T-cell leukemia; ATCC TIB-152) were preincubated with the following: no inhibitor (top and bottom left panels), 20 µM Z-LEHD-FMK (top and bottom center panels) or 20 µM control inhibitor, Z-FA-FMK (top and bottom right panels) for 30 min, and then either left untreated (bottom row) or treated with 4 µM of campthothecin for 3 hr (top row). Following incubation, cells were collected and stained with PE-Annexin V (Cat. No. 559763) to identify cells undergoing apoptosis. The results indicate that in campthothecin treated cells approximately 42% of the cells were induced to undergo apoptosis and the use of the caspase-9 inhibitor Z-LEHD-FMK reduced the level of apoptosis to half of that level observed in treated controls. Cells treated with Z-FA-FMK (Cat. No. 550411) showed similar results to the treated cells without inhibitor, indicating that the control inhibitor did not attenuate apoptosis.
      Z-LEHD-FMK, Caspase-9 Inhibitor
      Flow cytometric analysis of apoptosis in Jurkat cells.  Jurkat cells (Human T-cell leukemia; ATCC TIB-152) were preincubated with the following: no inhibitor (top and bottom left panels), 20 µM Z-LEHD-FMK (top and bottom center panels) or 20 µM control inhibitor, Z-FA-FMK (top and bottom right panels) for 30 min, and then either left untreated (bottom row) or treated with 4 µM of campthothecin for 3 hr (top row). Following incubation, cells were collected and stained with PE-Annexin V (Cat. No. 559763) to identify cells undergoing apoptosis. The results indicate that in campthothecin treated cells approximately 42% of the cells were induced to undergo apoptosis and the use of the caspase-9 inhibitor Z-LEHD-FMK reduced the level of apoptosis to half of that level observed in treated controls. Cells treated with Z-FA-FMK (Cat. No. 550411) showed similar results to the treated cells without inhibitor, indicating that the control inhibitor did not attenuate apoptosis.
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      BD Pharmingen™
      Flow cytometry (Routinely Tested)
      AB_2868883
      RUO


      Preparation And Storage

      Avoid multiple freeze-thaws of product.

      Z-LEHD-FMK, Caspase-9 Inhibitor is provided as a lyophilized powder. Store the lyophilized inhibitor at -20˚C. Reconstitute the inhibitor in DMSO before use. The reconstituted inhibitor may be stored in small aliquots at -20˚C.

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      Recommended Assay Procedures

      The Z-LEHD-FMK inhibitor can be used for both in vivo and in vitro cell based assays to measure the inhibition of apoptosis. While there are numerous methods to induce and measure apoptosis, Annexin V staining can be used on Jurkat cells (Human T-cell leukemia; ATCC TIB-152) for evaluating apoptosis (see figure).

      Reconstitute 1.0 mg of Z-LEHD-FMK in DMSO. A 10 mM stock solution may be prepared by dissolving 1.0 mg of Z-LEHD-FMK in 124 µl DMSO. When using the Z-LEHD-FMK inhibitor, the optimal concentration needed may vary with the experimental system being used and investigators are highly encouraged to titrate the reagent. As a precautionary note, please do not exceed a final DMSO concentration of 0.2% as higher levels may increase the risk for cellular toxicity which may mask the effect of the caspase inhibitor.

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      Product Notices

      1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
      2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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      550381 Rev. 4
      Antibody Details
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      Members of the caspase family play key roles in inflammation and mammalian apoptosis. Z-LEHD-FMK is an irreversible and cell permeable inhibitor of caspase-9. The peptide is O-methylated in the P1 position on aspartic acid providing enhanced stability and increased cell permeability. Z-LEHD-FMK can be used to inhibit primarily caspase-9 activity and to study events downsteam of caspase-9 activation. Z-LEHD-FMK has been reported to have a molecular weight of 804 Daltons.

      550381 Rev. 4
      Format Details
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      Purified
      Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
      Purified
      550381 Rev.4
      Citations & References
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      Development References (2)

      1. Maccarrone M, Lorenzon T, Bari M, Melino G, Finazzi-Agro A. Anandamide induces apoptosis in human cells via vanilloid receptors. Evidence for a protective role of cannabinoid receptors. J Biol Chem. 2000; 275(41):31938-31945. (Biology). View Reference
      2. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science. 1998; 281(5381):1312-1316. (Biology). View Reference
      550381 Rev. 4

      Please refer to Support Documents for Quality Certificates


      Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


      Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

      For Research Use Only. Not for use in diagnostic or therapeutic procedures.

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