Caspase, Annexin V, Cathepsin, Mitochondrial Membrane, Apo-BrdU™ TUNEL assay
Bio-Rad offers a quick and easy method for detecting apoptotic cells by flow cytometry with the Annexin V apoptosis kits.
Fig 1: Dot-plot showing Ramos Cells staining with Annexin V-FITC versus Propidium lodide.
Our wide range of Caspase FLICA™ apoptosis detection kits, allow analysis of active caspases in whole, living cells. The methodology of these caspase assay kits is based on a unique cell-permeable and non-cytotoxic reagent.
Fig 2: Results using Poly caspase FLICA™ kits (Product Code ICT091 & ICT092) analyzed by fluorescence microscopy show suspension cells labeled with FAM-VAD-FMK, and Hoechst 33342 stain. Both cells are apoptotic and dying - detected by (green) caspase activity and (blue) nuclear Hoechst staining. The left cell is much brighter green than the right cell, indicating that the left cell has more active caspases.
The JC-1, TMRE and TMRM MitoPT™ Kits allow clear differentiation between non-apoptotic and apoptotic cells through the simple and reproducible detection of the mitochondrial membrane permeability transition event.
Fig 3: Jurkat cells stained with MitoPT™ JC-1 (Product Code ICT943 & ICT944) and viewed through a fluorescence microscope. Non-apoptotic cells exhibit red stained mitochondria (2 left hand cells). Apoptotic cells at varying stages of mitochondrial membrane permeability appear green (3 right hand cells).
Bio-Rad’s Apo-BrdU™ TUNEL assay is based on the addition of labeled nucleotides to the free 3’-hydroxyl termini of double or single stranded DNA fragments. This method makes it easy to identify apoptotic cells since the DNA of healthy cells remains intact.
The TUNEL assay labels DNA fragments with bromolated deoxyuridine triphosphate nucleotides (Br-dUTP) through a reaction catalyzed by terminal deoxynucleotidyl transferase (TdT).
Fig 4. Flow cytometry data of APO-BRDUTM showing the log green fluorescence of positive control cells
We offer Magic Red™ Kits for analysis of active cathepsins in whole, living cells, utilizing a substrate based assay. These kits enable the detection of active cathepsins contained in lysosomes which are involved in the autophagy pathway, phagocytosis and when released into the cytoplasm can regulate both the apoptosis extrinsic and intrinsic pathways.
Fig5. Intracellular cathespin L activity detected in THP-1 cells using Magic Red™ Cathepsin L Kit (Products Code ICT941 / 942)
Visualize apoptosis events in real time with our pSIVA™ Real-Time Apoptosis Fluorescent Microscopy and Flow Cytometry Kits. pSIVA only fluoresces when bound to phosphatidylserine (PS), which becomes exposed at the outer plasma membrane as one of the early events of apoptosis. The pSIVA probe is conjugated to IANBD, a green emitting dye with spectral properties similar to FITC. No washing steps are required; simply add the probe to your cells and start visualizing apoptosis in real time. The pSIVA Real-Time Apoptosis Fluorescent Microscopy and Flow Cytometry Kits also contain the viability dye propidium iodide, allowing you to distinguish early apoptotic from late apoptotic and necrotic cells.
Fig. 6. HeLa cells were incubated A) in the presence or B) in the absence of staurosporine to induce apoptosis. pSIVA-IANBD and PI were added to the media and the cells imaged with the ZOE® Fluorescent Cell Imager. Early apoptotic cells are pSIVA-IANBD positive, shown in green, while late apoptotic cells are PI positive, shown in red.
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