Is Annexin V specific for Apoptosis?

Annexin-V is a specific PS-binding protein that can be used to detect apoptotic cells.

How is mitochondrial membrane potential detected?

Probes that detect mitochondrial membrane potential are positively charged, causing them to accumulate in the electronegative interior of the mitochondrion. Changes in the mitochondrial membrane potential can be measured by a variety of fluorescence techniques such as flow cytometry and fluorescence imaging.

What is mitochondrial membrane potential assay?

The Mitochondrial Membrane Potential Assay Kit (II) is a fluorescent assay that detects the mitochondrial membrane potential in living cells. TMRE is a cell membrane permeable, fluorescent dye that accumulates in intact mitochondria.

How do you test for mitochondrial function?

One option for assessing mitochondrial function is an organic acid test. This evaluates the metabolites from digestion, assimilation, metabolism, and the production of ATP. The preferred specimen is a urine collection; these metabolites are more easily extracted from urine than plasma.

How much is the mitochondrial membrane potential?

Mitochondrial membrane potential in cultured rat cortical neurons is −139 mV at rest. In response to electrical stimulation of the cells, it is regulated between −108 mV and −158 mV by concerted increases in energy demand and metabolic activation.

What is the typical potential difference across a mitochondrial membrane?

In a typical cell, the proton-motive force across the inner membrane of a respiring mitochondrion is about 200 mV and is made up of a membrane potential of about 140 mV and a pH gradient of about -1 pH unit.

Can you fix cells after annexin V staining?

Yes, cells can be fixed with formaldehyde after staining. Because Annexin V staining is dependent on calcium, all buffers used for washing and fixation should contain 1.25 mM CaCl2. Fixation may increase the background signal from Ethidium Homodimer III.

How are mitopt kits used to detect apoptosis?

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. An early indication of apoptosis involves a collapse in the electrochemical gradient across the mitochondrial membrane.

Which is the apoptosis assay for Jurkat cells?

Jurkat cells were stained using the FLISP Serine Protease Assay Kit ( APO009) and treated with staurosporine to induce apoptosis. Apoptotic cells positive for serine protease activity can be seen in the bottom right quadrant and dead cells positive for both serine protease and PI in the top right quadrant.

What kind of assay is used for mitochondria function?

(B) MitoProbe JC-1 Assay Kit was used to stain Jurkat cells (T-cell leukemia, human) which were then analyzed on a flow cytometer using 488 nm excitation with 530 nm and 585 nm bandpass emission filters. Green = apoptotic cells (reduced mitochondria membrane potential), red = normal cells.

How is mitochondrial depolarization indicated by a reagent?

Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. JC-1 Dye is available as a standalone reagent (Cat. No. T3168) or in the MitoProbe JC-1 Assay Kit which contains the JC-1 Dye, CCCP (a mitochondria membrane potential disrupter in DMSO), 10x PBS, and DMSO.

https://www.youtube.com/watch?v=7hScnHS9ylU

Is annexin V specific for apoptosis?

Annexin-V is a specific PS-binding protein that can be used to detect apoptotic cells.

What does annexin V positive mean?

apoptosis
Annexin V positive – PI negative populations represent cells in early apoptosis. > Annexin V positive – PI positive staining indicate cells are in necrosis (post-apoptotic necrosis or late apoptosis).

What does annexin V do?

Annexin A5 (or annexin V) is a cellular protein in the annexin group. In flow cytometry, annexin V is commonly used to detect apoptotic cells by its ability to bind to phosphatidylserine, a marker of apoptosis when it is on the outer leaflet of the plasma membrane.

Can annexin V be fixed?

In my experience, it is possible to fix annexin V stained cells and see great results on the flow. Stain cells with annexin V (in calcium-rich binding buffer). Wash cells in binding buffer to remove unbound AxV. Fix in your choice of fixative (diluted in binding buffer).

What Colour is annexin V?

green
Cells that have bound annexin V-FITC will show green staining in the plasma membrane. Cells that have lost membrane integrity will show red staining (PI) throughout the nucleus and a halo of green staining (FITC) on the cell surface (plasma membrane).

What triggers apoptosis?

Apoptosis can be activated by stimuli coming within the cell, including cell stressors, such as hypoxia or lack of nutrients, and agents that cause damage of DNA or other cell structures. A third pathway leading to apoptosis is specific of cytotoxic T lymphocytes (CTL) and natural killer cells (NK) (Chapter 30).

What does annexin V measure?

As a new method for detecting apoptosis, the Annexin V binding assay is based on the measurement of the loss of plasma membrane asymmetry.

What are the 4 stages of apoptosis?

Four Stages of Apoptosis Schematic To illustrate these apoptosis events and how to detect them, Bio-Rad has created a pathway which divides apoptosis into four stages: induction, early phase, mid phase and late phase (Figure 1).

What is the hallmark of apoptosis?

Apoptosis is characterised by a series of typical morphological features, such as shrinkage of the cell, fragmentation into membrane-bound apoptotic bodies and rapid phagocytosis by neighbouring cells.