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Mulitplex Caspase Activities

Detection of caspase 3/7 activities in jurkat cells.
Detection of caspase 3/7 activities in Jurkat cells. Jurkat cells were seeded on the same day at 200,000 cells /well/90 µL in a Costar black wall/clear bottom 96-well plate. The cells were treated with or without 1 µM staurosporine for 5 hours. The caspase 3/7 assay solution (100 µL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity was measured at Ex/Em = 540/620 nm.
Caspases, cysteine-aspartic proteases or cysteine-dependent aspartate directed proteases, are a family of cysteine proteases that play essential roles in apoptosis (programmed cell death), necrosis, and inflammation. Caspases are essential in cells for development and most other stages of adult life, and have been termed "executioner" proteins for their roles in cells. Some caspases are also required in the immune system for the maturation of lymphocytes. Failure of apoptosis is one of the main contributions to tumor development and autoimmune diseases. It is also reported that the unwanted apoptosis occurs with ischemia or Alzheimer's disease. The new findings have stimulated interest in caspases as potential therapeutic targets since they were discovered in the mid-1990s.

Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit is designed to monitor cell apoptosis through measuring Caspase 3 activation. Caspase 3 is widely accepted as a reliable indicator for cell apoptosis since the activation of caspase-3 (CPP32/apopain) is important for the initiation of apoptosis. Caspase 3 has substrate selectivity for the peptide sequence Asp-Glu-Val-Asp (DEVD). Z-DEVD-ProRed™ is used in the kit as the fluorogenic indicator for caspase-3 activity. Cleavage of ProRed™ DEVD blocking peptide residue by caspase 3 generates strongly red fluorescent ProRed™ that is fluorimetrically monitored at ~620 nm with excitation at ~530 nm.

Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit provides all the essential components with an optimized assay protocol. The assay is robust, and can be readily adapted for high-throughput assays in a wide variety of fluorescence platforms such as microplate assays. Using 100 µL of reagents per well in a 96-well format, this kit provides sufficient reagents to perform 100 assays. Using 25 µL of reagents per well in a 384-well format, this kit provides sufficient reagents to perform 400 assays.

Fluorescence spectra of AMC, R110, and ProRed
The fluoresence spectra of AMC, R110 and ProRed™ in aqueous buffer (pH 7.0). AMC, R110 and ProRed™ caspase substrates are well suited for multiplexing caspase activities.

Key Features of Cell Meter™ Caspase 3/7 Activity Assay Kits:

  • Multicolor capability, tricolor panel enables the simultaneous detection of multiple caspase activations (see Figure 1.5).
  • Robust, a mix and read format.
  • Convenient, compatible with common filter sets.

 

Table 1. Assay kits for measuring Caspase 3, 7, 8 and 9 activity

Kit
Platform
Ex (nm)
Em (nm)
Unit Size
Cat No.
Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Blue Fluorescence*Microplate, HCS360570200 tests22795
Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Green Fluorescence*Microplate, HCS490525200 tests22796
Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Red Fluorescence*Microplate, HCS540620200 tests22797
Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Green Fluorescence Optimized for Flow Cytometry*Flow Cytometer488530100 tests22823
Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Blue Fluorescence*Microplate, HCS370450200 tests22812
Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Green Fluorescence*Microplate, HCS490525200 tests22798
Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Red Fluorescence*Microplate, HCS540620100 tests22816
Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Blue Fluorescence*Microplate, HCS375435200 tests22813
Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Green Fluorescence*Microplate, HCS490525200 tests22798
Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Red Fluorescence*Microplate, HCS540620100 tests22816
Cell Meter™ Multiplexing Caspase 3/7, 8 and 9 Activity Assay Kit *Triple Fluorescence Colors*Microplate, HCSTriple FluorescenceTriple Fluorescence100 tests22820