How do you do TUNEL assay?
TUNEL assay methods
- a nucleotide directly conjugated to a fluorescent dye (usually FITC)
- a biotin-tagged nucleotides that is then bound by streptavidin-HRP and detected using a chromogenic HRP substrate, such as DAB, to generate a brown color.
What is the TUNEL assay used for?
Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay has been designed to detect apoptotic cells that undergo extensive DNA degradation during the late stages of apoptosis.
How long does a TUNEL assay take?
1–3 hours
Together, the TUNEL reaction mixture and the tissue section are incubated for 1–3 hours at 37°C, and protected from light to prevent the fluorescence from fading.
How do you quantify TUNEL staining?
In order to quantitate TUNEL assay results in tissue sections, manual counting of TUNEL-positive (TUNEL+) cells, ideally by two masked observers is performed. Observers count TUNEL+ cells and measure the respective retinal area, and results are expressed as either TUNEL+ cells/area or TUNEL+ cells/total cells.
Does TUNEL detect necrosis?
As pointed out by Loo almost a decade ago [67], “because TUNEL staining is nonspecific in the sense that the assay will label all free 3 -hydroxyl termini, irrespective of the molecular mechanisms that led to the development of these termini, TUNEL staining will also detect non-apoptotic cells—including necrotic …
How do you quantify apoptosis?
Two methods frequently used for quantifying apoptosis are counting morphologically positive cells on an H&E stain or evaluating the number of TUNEL-positive cells per 1000–2000 cells (4, 9, 16).
How long does a Tunel assay take?
How is BrdUTP detected in the APO-BrdU assay?
In the APO-BrdU assay, these ends are labeled with BrdUTP and terminal deoxynucleotidyl transferase (TdT) using the TUNEL technique. Once incorporated ino the DNA, BrdU is detected using a bright and photostable green-fluorescent Alexa Fluor™ 488 dye-labeled anti-BrdU antibody. View a selection guide for all apoptosis assays for flow cytometry.
What is the APO-BrdU™ kit?
The APO-BRDU™ Kit is a two color staining method for labeling DNA breaks and total cellular DNA to detect apoptotic cells by flow cytometry. Sufficient reagents are provided to process 60 cell suspensions.
What is the difference between the APO-direct™ and Apo-BrdU™ assays?
The APO-DIRECT™ assay (Cat. No. 51-6536KK) is a single-step method for labeling DNA breaks with FITC-dUTP, followed by flow cytometric analysis. The APO-BRDU™ Kit is a two color staining method for labeling DNA breaks and total cellular DNA to detect apoptotic cells by flow cytometry.
How much TDT is in the APO-BrdU™ kit?
One 0.045 ml bottle of TdT Enzyme [200 µg/ml (S.A.= 100,000 U/mg) in 50% (v/v) glycerol solution; will not freeze at -20°C] [Comp. No. 51-6581EZ] The APO-BRDU™ Kit consists of two parts (A and B). Part A (Comp. No. 51-6576AK) is shipped at ambient temperature and should be stored at 4°C upon arrival.
What can be detected in TUNEL assay?
The TUNEL assay is widely used for the detection of apoptotic events in paraffin-embedded tissue sections. The assay detects DNA fragmentation by nicking and when used in its improved form is claimed to be specific for DNA fragmentation occurring in cells in late apoptosis [314].
What is a TUNEL assay used for?
What does TUNEL stand for?
TUNEL is an acronym for terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling. Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that is normally active in primitive lymphoid cells to increase receptor diversity.
How do you know if a cell is apoptotic?
The morphologic criteria for identifying cells undergoing apoptosis are well established and include cytoplasmic condensation, loss of cell–cell contact, and cell shrinkage.
What do apoptotic cells look like?
A cell undergoing apoptosis shows a series of characteristic morphological changes. Early alterations include: Cell shrinkage and rounding occur because of the retraction lamellipodia and the breakdown of the proteinaceous cytoskeleton by caspases. The cytoplasm appears dense, and the organelles appear tightly packed.
Do apoptotic cells release cytokines?
While apoptosis facilitates the controlled degradation of intracellular proteins and organelles, pyroptosis and necroptosis lead to cell lysis and the release of a wide range of intracellular components and inflammatory cytokines.
What triggers cell apoptosis?
Apoptosis is mediated by proteolytic enzymes called caspases, which trigger cell death by cleaving specific proteins in the cytoplasm and nucleus. Caspases exist in all cells as inactive precursors, or procaspases, which are usually activated by cleavage by other caspases, producing a proteolytic caspase cascade.
Can the click-it TUNEL assay kits be used on tissue samples?
The original Click-iT TUNEL assay kits were optimized for cell culture samples and may also be used on tissue samples. We recommend using the Click-iT Plus TUNEL for In Situ Apoptosis Detection assay kits on tissue samples; the protocols for the Click-iT Plus TUNEL for In Situ Apoptosis Detection assay kits have been optimized for tissue.
How fast is the click-it Tunel imaging assay?
As a result, when compared to assays using other modified nucleotides, the Click-iT TUNEL imaging assay is fast (complete within 2 hours) and is able to detect a higher percentage of apoptotic cells under identical conditions (Figures 4–5). Furthermore, the Click-iT TUNEL assay allows multiplexing with surface and intracellular biomarker detection.
Can I use click-it plus Tunel for in situ apoptosis detection on tissue samples?
We recommend using the Click-iT Plus TUNEL for In Situ Apoptosis Detection assay kits on tissue samples; the protocols for the Click-iT Plus TUNEL for In Situ Apoptosis Detection assay kits have been optimized for tissue. The protocol was modified to improve accessibility of the TdT enzyme into the multiple cell layers of tissue samples.
Does click-it plus Tunel stain actin?
Additionally, the ability to stain actin using fluorescent-labeled phalloidin was also preserved. The Click-iT® Plus TUNEL assay contains all of the reagents needed to detect apoptotic cells from either tissue or cell samples.