DNA Fragmentation & Morphology

The later stages of apoptosis are characterized by changes in nuclear morphology, including DNA fragmentation, chromatin condensation, degradation of nuclear envelope, nuclear blebbing, and DNA strand breaks. We offer a variety of reagents for analyzing nuclear changes during apoptosis; many are compatible with other reagents and procedures used to detect additional cell health and viability parameters.

Also see nuclear assays for flow cytometry

Nuclear apoptosis assays

Cells undergoing apoptosis display an increase in nuclear chromatin condensation, which is visible using NucBlue® Live Cell Stain. Nuclear staining works for live or fixed cells and is readily combined with other detection procedures. DNA fragmentation that occurs during apoptosis produces DNA strand breaks, which can be analyzed using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end-labeling) assays. TUNEL assays using Click-iT® Plus technology can be used to study cultured cells and tissue sections, including FFPE-treated samples.

  • DNA stain to visualize chromatin condensation
  • TUNEL assays for imaging and HCS
  • TUNEL assays for tissue analysis

DNA stain to visualize chromatin condensation

DNA stains provide the fastest and most convenient route to distinguish the condensed nuclei in apoptotic cells. Nuclear staining can be performed on live or fixed cells, and we offer a choice of wavelengths for multiplexing with other cellular labels. The ReadyProbes® formulations are stable at room temperature and provided ready to use in convenient dropper bottles.  

image of cells stained with NucBlue® Live ReadyProbes® ReagentEndothelial cells stained with NucBlue® Live ReadyProbes® Reagent

TUNEL assays for imaging and HCS

For cultured cells, the Click-iT® TUNEL Alexa Fluor® Imaging Assays are optimized for HCS or microscope imaging. The small and specific labeling moiety enables a fast and efficient experimental protocol. The assay is complete within 2 hours, and provides efficient discrimination of even high levels of apoptotic cells in a population. With a choice of fluorescent labels, the Click-iT® TUNEL assay allows multiplexing with surface and intracellular biomarker detection.

  • Optimized for HCS
  • Efficient labeling protocol
  • Assay complete in 2 hours
  • Three wavelength options for multiplexing
  image of cells stained with Click-iT® TUNEL Alexa Fluor® 594 Imaging Assay reagents

HeLa cells were treated with staurosporine to induce apoptosis and stained using the Click-iT® TUNEL Alexa Fluor® 594 Imaging Assay, for Microscopy & HCS and Hoechst 33342. Images were acquired using a Thermo Scientific™ Cellomics® ArrayScan® VTI.

TUNEL assays for tissue analysis

The TUNEL assay is the most widely used method to detect DNA fragmented in apoptotic cells in tissue samples. Using a small labeling moiety and a bright fluorescent dye, Click-iT® Plus TUNEL assays have been demonstrated to detect apoptotic cells in several different FFPE (formalin-fixed, paraffin-embedded) tissue types. After the labeling moiety is incorporated into DNA fragments, detection is achieved through a “click” reaction using conditions mild enough to preserve the fluorescent signal from GFP or RFP and maintaining the ability to stain actin using fluorescently labeled phalloidin.

  • Optimized for tissue sections
  • Small labeling moiety for better penetration
  • Three wavelength options for multiplexing
  • Mild reaction, compatible with GFP and RFP

The Click-iT® Plus TUNEL assay generates a bright, photostable fluorescent signal in a choice of emission colors for multiplexing fluorescent dyes or proteins to enable a variety of experimental conditions.

  

image of mouse intestine section stained using Click-iT Plus TUNEL Assay reagentsFFPE sections of mouse intestine expressing GFP in epithelial cells were stained for apoptosis using the Click-iT® Plus TUNEL Assay with Alexa Fluor® 647. Alexa Fluor® 555 Phalloidin was used to stain actin, and nuclei were stained with NucBlue® Live ReadyProbes® Reagent.

Selection guide for nuclear apoptosis assays

 
NucBlue® Live ReadyProbes® Reagent
NucRed® Live 647 ReadyProbes® Reagent
NucBlue® Fixed Cell ReadyProbes® Reagent
Readout
Fluorescent staining of nucleic acids
Target
Membrane-permeable dyes targeting RNA and DNA
Membrane impermeable dyes target RNA and DNA
Common filter set
DAPI
Cy®5
DAPI
Labels
Hoechst 33342
NucRed® Live
DAPI
Ex/Em (nm)
360/460
638/686
360/460
Signal-to-noise ratio
Photostability
Multiplexing
Yes
Yes
Yes
Live cells
Yes
Yes
No
Fixed cells
No
No
Yes
Fixable
Yes
No
No
Format
6 x dropper bottles
6 x dropper bottles
6 x dropper bottles
Cat. No.
R37605
R37106
R37606
 
Click-iT® TUNEL Alexa Fluor® 488 Imaging Assay, for microscopy & HCS
Click-iT® TUNEL Alexa Fluor® 594 Imaging Assay, for microscopy & HCS
Click-iT® TUNEL Alexa Fluor® 647 Imaging Assay, for microscopy & HCS
Readout
Click-iT® TUNEL Assays for DNA fragmentation provide efficient discrimination of even high levels of apoptosis in a cell population.
Conjugated dye
Alexa Fluor® 488
Alexa Fluor® 594
Alexa Fluor® 647
Standard filter set
FITC
Texas Red®
Cy®5
Ex/Em (nm)
495/519
590/615
650/670
Bibliography
Citations
Fluorescence microscopy
Yes
HCS
Yes
HTS
No
Multiplexing
Yes
Live cells
No
Fixed cells
Mild fixation and permeabilization required
Fixable
Yes
Format
1 kit (1 microplate or 50 coverslips)
Cat. No.
C10245
C10246
C10247
 
Click-iT® Plus TUNEL for in situ apoptosis detection with Alexa Fluor® 488 dye
Click-iT® Plus TUNEL for in situ apoptosis detection with Alexa Fluor® 594 dye
Click-iT® Plus TUNEL for in situ apoptosis detection with Alexa Fluor® 647 dye
Readout
Click-iT® Plus TUNEL Assays for DNA fragmentation provide efficient discrimination of apoptosis in tissues with multiplexing capability for organic dyes and fluorescent proteins.
Conjugated dye
Alexa Fluor® 488
Alexa Fluor® 594
Alexa Fluor® 647
Standard filter set
FITC
Texas Red®
Cy®5
Ex/Em (nm)
495/519
590/615
650/670
Bibliography
Citations
Citations
Citations
Fluorescence microscopy
Yes
HCS
Yes
HTS
No
Multiplexing
Yes
Live cells
No
Fixed cells
Mild fixation and permeabilization required
Fixable
Yes
Format
1 kit/1 microplate or 50 coverslips
Cat. No.
C10617
C10618
C10619