235432 Caspase-3 Intracellular Activity Assay Kit II (PhiPhiLux® G₂D₂)

235432
  
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      Áttekintés

      Replacement Information

      Kulcsspecifikációk táblázata

      Detection Methods
      Fluorescence
      Description
      OverviewAssay kit for the determination by flow cytometry or fluorescence microscopy of caspase-3-like activity in intact cells undergoing apoptosis. Can be used to evaluate the ability of a test substance or drug candidate to activate or inhibit caspase-3. The assay is based on the reaction of the enzyme with the cell-permeable PhiPhiLux® G2D2 substrate. Each substrate molecule contains two fluorophores and the cleaved substrate fluoresces red.
      Catalogue Number235432
      Brand Family Calbiochem®
      Materials Required but Not Delivered 250 µl of fetal bovine serum (FCS).
      References
      ReferencesChang, S.H., et al. 2002. Exp. Cell Res. 277, 15.
      Kottke, T.J., et al. 2002. J. Biol. Chem. 277, 804.
      Liu, L., et al. 2002. Nat. Med. 8, 185.
      Telford, W.G., et al. 2002. Cytometry 47, 81.
      Packard, B.Z., et al. 2001. J. Immunol. 167, 5061.
      Finucane, D.M., et al. 1999. J. Biol. Chem. 274, 2225.
      Porter, A.G. and Janicke, R.U. 1999. Cell Death Differ. 6, 99.
      Robles, R., et al. 1999. Endocrinology 140, 2641.
      Hirata, H., et al. 1998. J. Exp. Med. 187, 587.
      Packard, B.Z., et al. 1998. J. Phys. Chem. B 102, 1820.
      Siegel, R.M., et al. 1998. J. Cell Biol. 141, 1243.
      Packard, B.Z., et al. 1997. Methods Enzymol. 278, 15.
      Nicholson, D.W., et al. 1995. Nature 376, 37.



      Product Information
      Detection methodFluorescence
      DeclarationSold under license of U.S. Patents 5,605,809 and 5,714,342. Not available for sale in Japan.
      Form30 Tests
      FormatFlow cytometry or fluorescence microscopy
      Kit containsFlow Cytometry Buffer, PhiPhiLux® G₂D₂ Substrate, and a user protocol.
      Applications
      Biological Information
      Assay time1.5-2 h
      Sample TypeIntact cells
      Physicochemical Information
      Emission max.
      Excitation max.
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Intracellular Caspase-3 Assay Kit allows is intended for studying the activation of caspase-3 and subsequent downstream events in whole cells. This kit can be used for either flow cytometry or fluorescence microscopy.
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Standard Handling
      Storage -20°C
      Storage ConditionsPrior to storage, centrifuge the reagent-containing vials briefly to remove any liquid from caps. Store kit at -20°C. Upon thawing, shake until the solution appears homogeneous. AVOID FREEZE / THAW CYCLES OF SOLUTIONS.
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsFlow Cytometry Buffer, PhiPhiLux® G₂D₂ Substrate, and a user protocol.
      Specifications

      Documentation

      Caspase-3 Intracellular Activity Assay Kit II (PhiPhiLux® G₂D₂) Certificates of Analysis

      TitleLot Number
      235432

      References

      Hivatkozások áttekintése
      Chang, S.H., et al. 2002. Exp. Cell Res. 277, 15.
      Kottke, T.J., et al. 2002. J. Biol. Chem. 277, 804.
      Liu, L., et al. 2002. Nat. Med. 8, 185.
      Telford, W.G., et al. 2002. Cytometry 47, 81.
      Packard, B.Z., et al. 2001. J. Immunol. 167, 5061.
      Finucane, D.M., et al. 1999. J. Biol. Chem. 274, 2225.
      Porter, A.G. and Janicke, R.U. 1999. Cell Death Differ. 6, 99.
      Robles, R., et al. 1999. Endocrinology 140, 2641.
      Hirata, H., et al. 1998. J. Exp. Med. 187, 587.
      Packard, B.Z., et al. 1998. J. Phys. Chem. B 102, 1820.
      Siegel, R.M., et al. 1998. J. Cell Biol. 141, 1243.
      Packard, B.Z., et al. 1997. Methods Enzymol. 278, 15.
      Nicholson, D.W., et al. 1995. Nature 376, 37.



      Brochure

      Title
      Caspases and other Apoptosis Related Tools Brochure
      User Protocol

      Revision23-September-2011 RFH
      Form30 Tests
      FormatFlow cytometry or fluorescence microscopy
      Detection methodFluorescence
      StoragePrior to storage, centrifuge the reagent-containing vials briefly to remove any liquid from caps. Store kit at -20°C. Upon thawing, shake until the solution appears homogeneous. AVOID FREEZE / THAW CYCLES OF SOLUTIONS.
      Intended useThe Intracellular Caspase-3 Assay Kit allows is intended for studying the activation of caspase-3 and subsequent downstream events in whole cells. This kit can be used for either flow cytometry or fluorescence microscopy.
      BackgroundCaspase-3 is one of the cysteine proteases most frequently activated during the process of programmed cell death. In response to pro-apoptotic stimuli, the 32 kDa proCaspase-3 is processed to an active enzyme consisting of two subunits of 17 and 12 kDa. This process is mediated by pathways either dependent or independent of mitochondrial cytochrome C release and Caspase-9 function. Activated caspase-3 is essential for the progression of apoptosis, resulting in the degradation of cellular proteins, apoptotic chromatin condensation, and DNA fragmentation.
      Principles of the assayPhiPhiLux™ is a peptide substrate for Caspase-3 that has been conjugated to two fluorophores (G₂D₂). The substrate contains the sequence GDEVDGI, with the caspase cleavage site underlined. The cleaved PhiPhiLux™G₂D₂ substrate has a red fluorescence with the following fluorescence peak characteristics: λex = 552 nm and λem = 580 nm. (The fluorescence of the uncleaved protease substrate is not completely quenched. This residual fluorescence can be used to monitor the cell loading of the substrate). When the folded peptide is cleaved, the fluorophores provide a high intensity fluorescent signal at a visible wavelength.
      Materials provided• 10 µM peptide substrate in RPMI 1640 medium with 25 mM HEPES: 4 vials, (at least 500 µl each)
      • flow cytometry dilution buffer: 1 bottle, (60 ml)
      Materials Required but not provided 250 µl of fetal bovine serum (FCS).
      Precautions and recommendations The cell density during incubation with the substrate can be as high as 0.5 to 1 million cells per sample. It is recommended that a control sample with the cells taken directly from a culture with cells in log phase be included in the assay to test for high cell density-induced apoptosis.
      While in certain settings one may be able to use the substrate at a concentration lower than 9 µM, it is not recommended. (Addition of FCS to a final concentration of 10% v/v would lower the substrate concentration to 9 µM.)
      Viable cell uptake of the substrate reaches a near maximum by 15 to 20 min at 37°C. As stated above, the incubation time may vary with cell type.
      It is recommend that in order to identify PI(-) apoptotic and uninduced cell populations, one analyze samples, first, without PI addition and then the same samples after PI addition. When one compares the dot plots of FL2 vs FL3 of these two samples, one can easily identify the PI(-) cell population as those cells that remain in the same location in the FL2 vs FL3 [FL2] dot plot after PI has been added. Place a gate around the PI(-) population and plot this gated FL1 histogram. This histogram should show both uninduced and apoptotic cells. One should avoid use of a threshold-based (orthogonal) gate for PI(+) cells, i.e., designation of cells above a certain FL2 or FL3 channel number as PI(+), since some very bright PI(-) apoptotic cells may appear in a region defined as PI(+). Be sure that the sample dilution volume is the same for all samples.
      If one observes the appearance of a very low fluorescence intensity population, i.e., lower than the uninduced cell population, then more than likely the samples have been overtreated with inducing agent or conditions that over induce the sample. In order to see the brighter apoptotic cell populations in histograms, these apoptotic cells must retain their membrane integrity. Please note that caspase-cleaved substrate fragments which generate positive signal in the PI(-) apoptotic cells are retained since the fragments diffuse through intact membranes significantly slower than the intact substrate. This differential diffusion rate will be lost when the cells loose their membrane integrity. When membrane integrity is lost, the cleaved substrate fragments leak out of such cells and these cells appear dark.
      Generally, cells in samples with a high percentage of PI(+) or TUNEL assay positive cells will be in late apoptosis and/or necrosis. Use of PhiPhiLux™ is optimal with samples containing a low percentage of PI(+) cells. In most cases we recommend lowering the inducing agent concentration rather than simply looking for an earlier time point. Appropriate time points for PhiPhiLux™ analysis of apoptotic cells should be such that a large percentage of viable PI(-) cells is present and the samples contain both uninduced cells plus caspase(+)/PI(-) cells. Examination of cells under a fluorescence microscope may assist in determining exact apoptosis inducing conditions.
      Detailed protocolApplication 1. Flow Cytometry Analysis

      Do not fix cells that have been previously incubated with the substrate for antibody labeling or labeling with other reagents. Remove all steps involving fixation or permeablization from your protocol.

      Incubation conditions:
      1. Treat target cells with chosen apoptosis-inducing reagent and/or inhibitor. (See Precautions and Recommendations)
      2. Aliquot cells into 1.5 to 2.0 ml microcentrifuge tubes, centrifuge and remove all of the culture medium to minimize substrate dilution. Use a gentle vacuum suction equipped with a capillary tip to completely remove the medium .
      3. To each of the cell pellets, add 50 to 75 µl of 10 µM substrate solution (add 5 µl of FCS, if 10% FCS is appropriate). The cell number in these solutions should be between 0.5 and 1 million per sample (See Precautions and Recommendations). Mix suspension containing cells and substrate by flicking tubes with finger tips. Do not vortex tubes containing cells, as apoptotic cells can be fragile.
      4. Incubate tubes in a 5% CO2 incubator at 37°C for 60 min before flow cytometric analysis. (See Precautions and Recommendations)
      5. Do not expose substrate solution to direct light or extremes of pH. Keep substrate at physiological pH during the experiment.
      6. Recommended flow cytometer setting: FL2 channel of a BD instrument with excitation at 488 nm. Use FL3 for propidium iodide (PI).
      7. Wash cells once by adding 1 ml of ice cold flow cytometry dilution buffer, centrifuging, and removing all buffer. Loosen the cell pellets by flicking the tubes with finger tips and then resuspend the loosened pellets in 1 ml of fresh dilution buffer. Do not vortex tubes containing cells. (See Precautions and Recommendations).
      8. Keep cell suspension on ice until analysis by flow cytometry.
      9. All samples should be analyzed within 60 to 90 min of completion of the 37°C incubation.
      10. After collecting data by the preceding procedure, one can delete PI(+) cells if a drop of a 5-10 µg/ml PI solution is added and samples are rerun on the flow cytometer (final PI concentration of @ 100 ng/ml). Reanalysis should be within 5 min of PI addition. Alternatively, if an additional 2.0 ml of flow cytometry dilution buffer is added to each sample, then cells that would be PI(+) will shift to a lower intensity range due to the leakage of intact PhiPhiLux™G1D2 substrate. Thus, the fluorescence of dead cells, i.e., those that would be PI(+), will show lower fluorescence intensity in the FL2 channel than will apoptotic PI(-) or non-apoptotic cells. (See Precautions and Recommendations).

      Application 2. Fluorescence Microscopy

      Incubation conditions:
      Do not fix cells that have been previously incubated with the substrate for antibody labeling or labeling with other reagents. Remove all steps involving fixation or permeabilization.

      1. Treat target cells with chosen apoptosis-inducing reagent and/or inhibitor. (See Precautions and Recommendations)
      2. (a) For suspension cells, aliquot cells into 1.5 to 2.0 ml microcentrifuge tubes, then centrifuge and remove as much the culture medium as possible to minimize the substrate dilution. (b) For adherent cells, remove all culture medium.
      3. (a) For suspension cells, to each of the centrifuged cell pellets, add 50 to 75 µl of 10 µM substrate solution (add 5 µl of FCS, if 10% FCS is appropriate). The cell number in these solutions should be between 0.5 and 1 million per sample (See Precautions and Recommendations). Mix suspension containing cells and substrate by flicking tubes with finger tips. Do not vortex tubes containing cells. (b) For adherent cells, add enough substrate solution to completely cover the monolayer or individual adherent cells. With either configuration be sure to remove all medium before addition of substrate-containing solution to minimize dilution of substrate.
      4. Incubate suspension cell samples in open tubes or adherent cells in a 5% CO2 incubator at 37°C for 30-60 min. Exact incubation times are cell type and inducer specific. For some cell types a concentration lower than 10 µM may be appropriate.
      5. Do not expose substrate to direct light or exposure to extremes of pH. Addition of HEPES buffer pH 7.4 to a final concentration of 20 mM will help maintain substrate at physiological pH during the experiment.
      6. Immediately following incubation with the PhiPhiLux™G2D2 substrate: (a) For suspension cells dilute with 1.0 ml physiological buffer, e.g., PBS, at 4°C centrifuge, and replace the supernatant with 1.0 ml of fresh buffer. Repeat this cell washing process two to four more times depending on your fluorescence microscope's lamp power. Check cells after each wash under fluorescence microscope to see if the background fluorescence is dark enough to distinguish between untreated control and treated cells. Recommended microscopy settings are rhodamine filters. (b) For adherent cells remove PhiPhiLux™G2D2 substrate-containing medium and wash gently with buffer. Perform a similar number of cell washing cycles as suspension cell samples. After each cycle make observation under fluorescence microscope to evaluate the background fluorescence level. Take special care in washing adherent cells since apoptotic cells are generally more easily detached from the plate than non-apoptotic cells.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      PhiPhiLux™G₂D₂ is a registered trademark of Oncolmmunin
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.