Ex/Em (nm) 556/579
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Ex/Em (nm)556/579MWN/ACAS #N/ASolventN/AStorageF/D/LCategoryCell Analysis
RelatedApoptosis and Cytotoxicity
DNA fragmentation represents a characteristic of late stage apoptosis. DNA fragmentation in apoptotic cells can be detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). The TUNEL assay relies on the presence of nicks in the DNA which can be identified by TdT, an enzyme that catalyzes the addition of dUTPs that are secondarily labeled with a marker. All the existing TUNEL assays contain the highly toxic sodium cacodylate which might induces apoptosis and also decrease DNA production and DNA strands. Our Cell Meter™ TUNEL Apoptosis Assay Kit uses proprietary buffer system free of sodium cacodylate. The kit is based on incorporation of a fluorescence dye TF3 modified deoxyuridine 5-triphosphates (TF3-dUTP) at the 3 OH ends of the DNA fragments that form during apoptosis. The assay is optimized for the direct detection of apoptosis in either detached or attached cells without using antibody. The kit provides all the essential components with an optimized assay protocol. It is suitable for fluorescence microplate reader, fluorescence microscope, or flow cytometer. Its signal can be easily detected at Ex/Em = 550nm/590 nm.
This protocol only provides a guideline, and should be modified according to your specific needs.
Note: Thaw Components C at room temperature, keep Components A and B on ice before use.
1. Culture cells to an optimal density for apoptosis induction according to your specific protocol. We recommend about 30,000 to 50,000 cells/well for adherent cells grown in a 96-well microplate culture, or about 1 to 2 x 106 cells/mL for non-adherent cells. At the same time, culture a non-induced negative control cell population at the same density as the induced population for every labeling condition. Here are a few examples for inducing apoptosis in suspension culture:
1) Treat Jurkat cells with 2 μg/ml camptothecin for 3 hours.
2) Treat Jurkat cells with 1 μM staurosporine for 3 hours.
3) Treat HL-60 cells with 4 μg/ml camptothecin for 4 hours.
4) Treat HL-60 cells with 1 μM staurosporine for 4 hours.
2. Fixation and Permeabilization
2.1 Remove cell media.
2.2 Add 100 μL/well/96-well plate of 4% formaldehyde fixative buffer (not supplied) to each well.
Note: For non-adherent cells, add desired amount (such as 2 x 106 cells/mL) of 4% formaldehyde fixative buffer.
2.3 Incubate plates for 20 to 30 minutes at room temperature.
2.4 Remove fixative.
Optional: add 100 μL/well/96-well plate of the permeabilization reagent (0.2% Triton X-100 in PBS, not supplied) after the fixation if needed, and incubate the plate for 10 minutes at room temperature.
2.5 Wash the cells with PBS 2-3 times.
Optional: You may also prepare a positive control for TUNEL reaction using DNAase I by digesting cells with DNAase I for 30 min at room temperature before proceed to TUNEL reaction (Step 3)
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