FAM-Spacer-Leu-CMK Green FLISP Assay Kit | FAM-Spacer-Leu-CMK assay kit

FAM-Spacer-Leu-CMK Green FLISP Assay Kit

Cat. Num. AAA258003

• Synonyms: FAM-Spacer-Leu-CMK Green FLISP; FAM-Spacer-Leu-CMK Green FLISP Assay Kit; FAM-Spacer-Leu-CMK assay kit

• Reagents: FSLCK
• Sample Protocol: Culture cells up to 1 x 10^6 cells/mL. Follow your experimental protocol where chymotrypsin-like enzyme activity will be investigated; create positive and negative controls for comparing chymotrypsin activity. Reconstitute the vial of FLISP reagent with the appropriate volume of DMSO to form the stock concentrate (can be frozen for future use). Dilute the stock concentrate with PBS to form the 50X sample spiking solution. Add 6-10 uL of the FLISP spiking solution directly to a 300-500 uL aliquot of your cell culture for labeling. Incubate 30 minutes -1 hour at 37 degree C in the dark. Wash cells 3X with fresh media or 1x working dilution of FLISP wash buffer. If desired, label cells with Hoechst stain. If desired, label cells with Propidium Iodide. If desired, fix cells for future analysis. Analyze data using a fluorescence microscope, plate reader, or flow cytometer.
• Target: Chymotrypsin-like enzymes
• Excitation / Emission: 488 nm / 530 nm
• Method of Analysis: Flow Cytometer, Fluorescence Microscope, Fluorescence Plate Reader
• Types of Samples: Cell Culture, Tissue
• Preparation and Storage: FLISP at -20 degree C; Other Kit Components at 2 - 8 degree C

Testing Data

(HL-60 cells were labeled with FAM-FLISP and ICT's SR-VAD-FMK FLICA caspase reagent (kit#917) after incubation with camptothecin for 3hours. Cells with active serine proteases staingreen (FAM-FLISP, y-axis, Fig. 1) and cellswith active caspases stain red (SR-VAD-FMK,x-axis, Fig. 5). Co-localization of serineprotease activity versus caspase activity isevident in dually stained cells (Figs. 2, 3, 4, &6).The DIC image (Fig. 7) reveals many negativecells (Dr. Zbigniew Darzynkiewicz, BranderCancer Center). )

Related Product Information for FAM-Spacer-Leu-CMK assay kit

Description: Detect changes in intracellular chymotrypsin-like serine protease enzyme activity in whole living cells with the FLISP, Fluorescent-Labeled Inhibitors of Serine Proteases, product line of assay kits. This assay utilizes a reagent (FSLCK) containing a leucine (L) chymotrypsin-targeting amino acid residue linked on the amino terminus with a carboxyfluorescein (FAM) fluorescent reporter tag. The carboxyl end of the L amino acid residue contains a reactive chloromethyl ketone (CMK) group that targets the catalytic site of serine proteases. After quickly penetrating the lipid bilayer membranes of the target cell population, the chymotrypsin-targeting FLISP probe FSLCK will interact with the active catalytic sites of chymotrypsin-like proteases, quickly forming covalent bonds with the reactive site histidine (N-H). Unbound FLISP reagent is easily removed during the wash step, resulting in cells with greater quantities of active chymotrypsin-like enzyme activity showing a greater fluorescence potential than cells that did not undergo an upregulation of serine protease activity. Green FAM-FLISP probes like FSLCK are excited at 488 nm and emit in the green wavelength range of 525 nm. Red fluorescence FLISP probes are also available. FLISP probes are cell permeant and non-cytotoxic at the concentrations suggested in the assay protocol. The FAM-spacer-L-CMK chymotrypsin enzyme detection assay kits can be used in conjunction with existing apoptosis detection protocols. Each FLISP FSLCK kit includes either 25 tests or 100 tests of FAM-spacer-F-CMK reagent, 10X wash buffer, 10X fixative, PI vital stain, and Hoechst 33342 dye. FSFCK-stained cells can be analyzed using flow cytometry, fluorescence microscopy, and fluorescence plate reader evaluation methodology.

Background: Chymotrypsin-like enzymes cleave their substrate proteins at the carboxy terminal end of amino acids containing hydrophobic aliphatic or aromatic R-group side chains such as those found on leucine and phenylalanine amino acid structures [1]. The FSLCK FLISP probe, consisting of FAM-spacer-Leu-CMK, is identical to the FAM-Leu-CMK probe except FAM-spacer-Leu-CMK includes an additional six carbon spacer group to minimize possible steric hindrance issues. Both the FAM-spacer-Leu-CMK and FAM-Leu-CMK probes are fluorescent labeled analogs of the early chymotrypsin-like enzyme inhibitor N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) [2]. When the two versions of this FAM-Leu-CMK probe (either with or without the six carbon spacer option) were compared in staurosporine induction experiments in Jurkat cells, there were no detectable differences in the chymotrypsin-like enzyme labeling pattern between the two forms of the leucine containing FLISP probe.. Early studies using topoisomerase inhibitors in HL-60 cells indicated that TPCK, N-tosyl-L-lysine chloromethyl ketone (TLCK) and other serine protease inhibitors were able to prevent internucleosomal DNA degradation associated with apoptosis [3]. Camptothecin induction experiments using HL-60 cells indicated a high linear correlation (r = 0.98) between apoptosis induction as measured by FAM-VAD-FMK binding to cells and FLISP probe binding frequency [4]. Dual staining experiments were also run using FAM-Phe-CMK or FAM-Leu-CMK (chymotrypsin detection probes) and our red FLICA poly caspase detection probe [4-5]. This comparison also showed a correlation between green fluorescence intensity staining from FAM-Phe-CMK (r = 0.72) or FAM-Leu-CMK (r = 0.82) stained cells and red FLICA-stained cells. This group showed that the chymotrypsin-like enzyme inhibitor, TPCK, had a strong suppressive effect on caspase activation and apoptosis induction. In contrast, the trypsin-like enzyme inhibitor, TLCK, had very little effect on caspase activation events as measured by the binding of the red FLICA probe in camptothecin-induced cell populations [4].

Product Categories/Family for FAM-Spacer-Leu-CMK assay kit

Apoptosis Assays/Serine Protease Detection

References

Czapinska, H. and J. Otlewski. 1999. Structural and energetic determinants of the S1-site specificity in serine proteases. Eur. J. Biochem. 260: 571-595. Grabarek, J., M. Dragan, B.W. Lee, G.L. Johnson, and Z. Darzynkiewicz. 2002. Activation of chymotrypsin-like serine proteases during apoptosis detected by affinity-labeling of the enzymatic center with fluoresceinated inhibitor. Int. J. Oncol. 20: 225-233. Zhu, H., D. Dinsdale, E.S. Alnemri, and G.M. Cohen. 1997. Apoptosis in human monocytic THP-1 cells involves several distinct targets of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK). Cell Death Diff. 4: 590-599. Grabarek, J., L. Du, G.L. Johnson, B.W. Lee, D. J. Phelps, and Z. Darzynkiewicz. 2002. Sequential activation of caspases and serine proteases (Serpases). Cell Cycle 1: 124-131. Grabarek, J., M. Dragan, B.W. Lee, G.L. Johnson, and Z. Darzynkiewicz. 2002. Activation of chymotrypsin-like serine protease(s) during apoptosis detected by affinity-labeling of the enzymatic center with fluoresceinated inhibitor. Int. J. Oncol. 20: 225-233.