Dual Sensor: DNA Damage & Caspase 3 Detection

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Fluoro ssDNA Caspase 3™ - DNACasp^TM
A Dual antibody assay to simultaneously detect DNA Damage (Single Stranded) & active Caspase 3 in apoptotic cells

Key Benefits:

Much more Reliable as compared to TUNEL assays - No False positive signals
Multiple parameter detection of DNA damage and caspase 3 activity simultaneously
Readout - Flow cytometry, Fluorescence 96 well plate reader, Fluorescence microscope
Yields both quantitative and qualitative results. Gives a strong positive signal.
Ease Of Use: Dual parameter results to confirm apoptosis in cells.
Compatible with human, mouse, rat, bovine, porcine species

Introduction:

1. Antibody to Single Stranded DNA

A widely used cytochemical technique for evaluation of DNA damage associated with apoptosis is the terminal deoxynucleotidyl transferase-mediated in situ end labeling or TUNEL assay. However the TUNEL assay has its drawbacks in that false positive staining makes the assay unreliable as a marker for apoptosis ^1-5. A more universal and specific marker for detecting apoptosis associated DNA damage is to measure the morphological changes in nuclei that reflect chromatin condensation into compact masses. ^6-7. Further biochemical and cytochemical studies have demonstrated the increased susceptibility of apoptotic DNA to thermal denaturation. Analysis of nuclei by scanning calorimetry to detect thermal induced DNA denaturation and analysis of DNA fragmentation by electrophoresis have shown that intact apoptotic DNA is susceptible to denaturation at lower temperatures then that of non-apoptotic cells ⁸.

2. Antibody to Active Caspase 3

Apoptosis is an evolutionarily conserved form of cell suicide, which follows a specialized cellular process. The central component of this process is a cascade of proteolytic enzymes called caspases. These enzymes participate in a series of reactions that are triggered in response to pro-apoptotic signals and result in cleavage of protein substrates, causing the disassembly of the cell ¹³. Caspases have been identified in organisms ranging from C. elegans to humans. The mammalian caspases play distinct roles in apoptosis and inflammation. In apoptosis, caspases are responsible for proteolytic cleavages that lead to cell disassembly (effector caspases), and are involved in upstream regulatory events (initiator caspases). An active caspase consists of two large (~20 kD) and two small (~10 kD) subunits to form two heterodimers which associate in a tetramer ^14-16. As is common with other proteases, caspases are synthesized as precursors that undergo proteolytic maturation, either autocatalytically or in a cascade by enzymes with similar specificity ¹⁷. Caspase 3, also known as CPP-32, Apopain or Yama, is a key effector caspase in the apoptotic pathway ¹⁸. It is present in many different cell lineages and is responsible for the cleavage of a variety of molecules such as poly ADP-ribose polymerase (PARP), protein kinase Cd, actin and DNA-dependent protein kinase ^19-20.

Assay Principle:

Cell Technology introduces a dual parameter antibody based assay to detect DNA damage (heat denatured single stranded DNA: ssDNA)^9-12 and active caspase 3 in apoptotic cells. The assay utilizes a monoclonal antibody generated against ssDNA and a primary rabbit affinity purified polyclonal antibody raised against amino acid 163-175 of murine caspase 3 ¹². This neo epitope is present on the p18 subunit of cleaved caspase 3 ²¹.

DNA Damage detection: Excitation: 488nm, Emission: 530nm (FL1)

Caspase 3 detection: Excitation: 633nm, Emission: 670nm (LP) (FL4)

Kit contents and Long Term storage:

1. Mouse anti single stranded DNA………………… Part 1003 -70°C aliquot

2. Rabbit anti active Caspase 3 ………………… Part 1006 4-8°C

3. Goat Anti mouse FITC labeled………… Part 2006 4-8°C

4. Goat Anti Rabbit APC ………… Part 2007 4-8°C

5. Fixative…………………………..…………………………… Part 3033 -20°C

6. 10 X Wash Buffer…………………………………………… Part 3037 -20°C

7. 1X Denaturing Buffer ……………………………Part 3036 Room Temp

The following kits are available:
Catalog	Size	Price
DNACasp3-1	25 Tests	€325
DNACasp3-2	50 Tests	€625

(A)

(B)

Fig (A): Negative control: Jurkat cells treated with DMSO for 3 hours and Fig (B): Positive Control: Jurkat cells treated with 1 mM stauropsorine for 3 hours. The cells were stained with Fluoro ssDNA Caspase 3 kit as described in the protocol. Key: FL 1-H = Anti active ssDNA stain (DNA damage), FL 4-H = anti active caspase 3 stain.