image of apoptosis detected in cells using specific stainsThe 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

Hoechst 33342 is perhaps the most ubiquitous nuclear fluorescence stain for live and fixed cells and tissue sections. The cell-permeant stain binds preferentially to AT-rich regions of DNA. It is excited by UV light and emits blue fluorescence at 460 nm when bound to DNA. The spectral properties of Hoechst 33342 and its large Stokes shift make it ideal for use with green and red fluorophores in multicolor experiments. Because of its high affinity for DNA, Hoechst 33342 is frequently used in cell replication studies to distinguish condensed nuclei in apoptotic cells. Formulated as NucBlue® Live ReadyProbes® Reagent, this classic stain is room temperature–stable and ready to use in a convenient dropper bottle.  

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

Changes in DNA morphology such as nuclear condensation or DNA fragmentation are efficient indicators of apoptosis.

 
Readout NucBlue® Live Cell Stain provides a simple indicator of nuclear condensation easily multiplexed with other apoptosis markers
Conjugated dye Hoechst 33342
Standard filter set DAPI
Ex/Em (nm) 350/461
Bibliography Citations
Fluorescence microscopy Yes
HCS Yes
HTS No
Multiplexing Yes
Live cells Yes
Fixed cells Yes
Fixable Yes
Format 1 kit
Cat. No. R37605
 
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
 
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