Nuclear Apoptosis Assays for Flow Cytometry

Various 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 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.

Analysis of chromatin condensation with Vybrant DyeCycle Violet stain and SYTOX AADvanced 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.

Condensed chromatin stainDead cell stainLasersEx/EmCat. No.
Hoechst 33342PIUV and 488 nmHoechst 33342: 350/461 nm
PI: 535/617 nm
V13244
YO-PRO-1
Hoechst 33342
PIUV and 488 nmHoechst 33342: 350/461 nm
YO-PRO-1: 491/509 nm
PI: 535/617 nm
V23201
Vybrant DyeCycle Violet stainSYTOX AADvanced405 and 488 nmVybrant DyeCycle Violet stain: 369/437 nm
SYTOX AADvanced: 546/647 nm
A35135
YO-PRO-1PI488 nmYO-PRO-1: 491/509 nm
PI: 535/617 nm
V13243
PO-PRO-17-AADUV and 488 nmPO-PRO-1: 434/456 nm
7-AAD: 546/647 nm
V35123
Condensed chromatin stainDead cell stainLasersEx/EmCat. No.
Hoechst 33342PIUV and 488 nmHoechst 33342: 350/461 nm
PI: 535/617 nm
V13244
YO-PRO-1
Hoechst 33342
PIUV and 488 nmHoechst 33342: 350/461 nm
YO-PRO-1: 491/509 nm
PI: 535/617 nm
V23201
Vybrant DyeCycle Violet stainSYTOX AADvanced405 and 488 nmVybrant DyeCycle Violet stain: 369/437 nm
SYTOX AADvanced: 546/647 nm
A35135
YO-PRO-1PI488 nmYO-PRO-1: 491/509 nm
PI: 535/617 nm
V13243
PO-PRO-17-AADUV and 488 nmPO-PRO-1: 434/456 nm
7-AAD: 546/647 nm
V35123

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.

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