Nuclear Apoptosis Assays for Flow Cytometry
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. Invitrogen offers a variety of unique reagents for analyzing nuclear changes during apoptosis; many are compatible with reagents used to detect additional apoptotic parameters using flow cytometry.
Nuclear Chromatin Condensation
Cells undergoing apoptosis display an increase in nuclear chromatin condensation. As the chromatin condenses, cell-permeable nucleic acid stains becomes hyperfluorescent, thus enabling the identification of apoptotic cells when combined with a traditional dead-cell stain.
Figure 1. Analysis of chromatin condensation with Vybrant® DyeCycle™ Violet stain and SYTOX® AADvanced™ stain. Jurkat cells (T-cell leukemia, human) were treated with 10 µM camptothecin for 6 hours (3B) or left untreated as a control (3A). Cells were then mixed with the reagents in the Apoptosis Kit- Vybrant® DyeCycle™ Violet/SYTOX® AADvanced™ Kit and analyzed by flow cytometry using 405/488 nm dual excitation. Note that the campthothecin-treated cells (3B) have a higher percentage of apoptotic cells than the basal level of apoptosis seen in the control cells (3A). A=apoptotic cells, V = viable cells, N = necrotic cells.
|Hoechst 33342||UV||350/461||H1399||product details|
|Vybrant® DyeCycle™ Violet||405||369/437||V35003||product details|
|YO-PRO®-1 iodide||488||491/509||Y3603||product details|
DNA Fragmentation (TUNEL Assay)
DNA fragmentation that occurs during apoptosis produces DNA strand breaks, and can be analyzed using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays. The APO-BrdU TUNEL assay is a two-color assay for labeling DNA breaks and total cellular DNA to detect apoptotic cells by imaging or flow cytometry. Propidium iodide is also included to analyze total DNA content or cell-cycle phase.
|APO-BrdU TUNEL Assay Kit with Alexa Fluor® 488 anti-BrdU||488||495/519||A23210||product details|