Se ha denunciado esta presentación.
Utilizamos tu perfil de LinkedIn y tus datos de actividad para personalizar los anuncios y mostrarte publicidad más relevante. Puedes cambiar tus preferencias de publicidad en cualquier momento.
Cell viability Assays
Sanju kaladharan
Viability assays
• Cell Viability Assay is a homogeneous method
to determine the number of viable cells in
culture.
• Cell...
• Cell viability and cytotoxicity assays are used for
drug screening and cytotoxicity tests of
chemicals.
They are based o...
DNA synthesis cell proliferation assays
• One of the most reliable and accurate assay types
is measurement of DNA synthesi...
Metabolic cell proliferation assays
• Another measure of cell proliferation is the metabolic activity of a
population of c...
• Four types of tetrazolium salts are most common:MTT,XTT MTS and WST1.
• A disadvantage of MTT is that it is insoluble in...
Tetrazolium Reduction Assays
• The most commonly used compounds include: MTT, MTS, XTT, and WST-1.
These compounds fall in...
MTT Tetrazolium Assay Concept
• This is a colorimetric assay that measures the reduction of yellow 3-(4,5-
dimethythiazol2...
Measuring [ATP]
• It takes advantage of the tight regulation of intracellular ATP within
cells.
• Dying or dead cells cont...
• ATP has been widely accepted as a valid marker of viable cells. When cells
lose membrane integrity, they lose the abilit...
Sulforhodamine B Cell Cytotoxicity Assay
• This assay relies on the ability of SRB to bind cellular protein
components and...
Plasma membrane integrity
• Assessing cell membrane integrity is one of the most common and
straightforward ways to measur...
Cell viability assays
Cell viability assays
Cell viability assays
Cell viability assays
Cell viability assays
Cell viability assays
Cell viability assays
Cell viability assays
Próxima SlideShare
Cargando en…5
×

Cell viability assays

CELL VIABILITY ASSAYS

  • Sé el primero en comentar

Cell viability assays

  1. 1. Cell viability Assays Sanju kaladharan
  2. 2. Viability assays • Cell Viability Assay is a homogeneous method to determine the number of viable cells in culture. • Cell viability, defined as the number of healthy cells in a sample, determines the amount of cells (regardless of phase around the cell cycle) that are living or dead, based on a total cell sample.
  3. 3. • Cell viability and cytotoxicity assays are used for drug screening and cytotoxicity tests of chemicals. They are based on various cell functions such as • enzyme activity, • cell membrane permeability, • cell adherence, ATP production, • co-enzyme production, and • nucleotide uptake activity
  4. 4. DNA synthesis cell proliferation assays • One of the most reliable and accurate assay types is measurement of DNA synthesized in the presence of a label. • Traditional cell proliferation assays involve incubating cells for a few hours to overnight with 3H-thymidine. • Proliferating cells incorporate the radioactive label into their nascent DNA, which can be washed, adhered to filters and then measured using a scintillation counter.
  5. 5. Metabolic cell proliferation assays • Another measure of cell proliferation is the metabolic activity of a population of cells. • Tetrazolium salts or Alamar Blue are compounds that become reduced in the environment of metabolically active cells, forming a formazan dye that subsequently changes the color of the media. • This is caused by increased activity of the enzyme lactate dehydrogenase during proliferation. • The absorption of the media-containing dye solution can be read using a spectrophotometer or microplate reader in low- or high-throughput configurations.
  6. 6. • Four types of tetrazolium salts are most common:MTT,XTT MTS and WST1. • A disadvantage of MTT is that it is insoluble in standard culture medium, and the formazan crystals produced during reduction must be dissolved in DMSO or isopropanol. • Because of this, MTT is mainly an endpoint assay. The other salts, as well as Alamar Blue, are soluble in culture media and are nontoxic. • They can be used for continuous monitoring, to follow dynamic changes in proliferation over time. • XTT reduces less efficiently and may need additional factors added. WST1 is more sensitive, reduces more efficiently and shows faster color development compared to the other salts. • Alamar Blue is also sensitive, capable of detecting as few as 100 cells in a well of a microtiter plate. • The tetrazolium salts and Alamar Blue redox dyes can be quantified with a range of instruments for conventional or high-throughput studies using, for example, standard spectrophotometers or spectrofluorometers or plate readers for spectrophotometric or spectrofluorometric microtiter well plates.
  7. 7. Tetrazolium Reduction Assays • The most commonly used compounds include: MTT, MTS, XTT, and WST-1. These compounds fall into two basic categories: • 1) MTT which is positively charged and readily penetrates viable eukaryotic cells and • 2) those such as MTS, XTT, and WST-1 which are negatively charged and do not readily penetrate cells. • The latter class (MTS, XTT, WST-1) are typically used with an intermediate electron acceptor that can transfer electrons from the cytoplasm or plasma membrane to facilitate the reduction of the tetrazolium into the colored formazan product.
  8. 8. MTT Tetrazolium Assay Concept • This is a colorimetric assay that measures the reduction of yellow 3-(4,5- dimethythiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. • The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured (dark purple) formazan product. • The cells are then solubilised with an organic solvent (eg. isopropanol) and the released, solubilised formazan reagent is measured spectrophotometrically. • Since reduction of MTT can only occur in metabolically active cells the level of activity is a measure of the viability of the cells
  9. 9. Measuring [ATP] • It takes advantage of the tight regulation of intracellular ATP within cells. • Dying or dead cells contain little to no ATP, so there is a tight linear relationship between cell number and the concentration of ATP measured in a cell lysate or extract. • The bioluminescence-based detection of ATP, using the enzyme luciferase and its substrate luciferin, provides a very sensitive readout. • In the presence of ATP, luciferase produces light (proportional to the ATP concentration) that can be detected by a luminometer or any microplate reader capable of reading luminescent signals. • This approach is also well suited to high-throughput cell proliferation assays and screening.
  10. 10. • ATP has been widely accepted as a valid marker of viable cells. When cells lose membrane integrity, they lose the ability to synthesize ATP and endogenous ATPases rapidly deplete any remaining ATP from the cytoplasm. • The ATP detection reagent contains detergent to lyse the cells, ATPase inhibitors to stabilize the ATP that is released from the lysed cells, luciferin as a substrate, and the stable form of luciferase to catalyze the reaction that generates photons of light.
  11. 11. Sulforhodamine B Cell Cytotoxicity Assay • This assay relies on the ability of SRB to bind cellular protein components and measure the total biomass. • SRB is a bright-pink aminoxanthene dye that can form an electrostatic complex with basic amino acid residues of proteins in slightly acidic conditions but it can dissociate under basic conditions. • It has been widely used for drug toxicity screening against different types of cancerous and non-cancerous cell lines. • In addition, this assay is independent of cell metabolic activity and therefore should show less interference by the testing compounds. • Since the binding of SRB is stoichiometric, the incorporated dye released from stained cells after washing is directly proportional to the cell biomass and can be measured at 565 nm
  12. 12. Plasma membrane integrity • Assessing cell membrane integrity is one of the most common and straightforward ways to measure cell viability and assess cytotoxic consequences. Compounds that have cytotoxic effects often compromise cell membrane integrity and induce necrosis. • Dyes, such as propidium iodide and 7-AAD, are normally excluded from the inside of healthy cells; however, if the cell membrane has been compromised, they freely cross the membrane and stain intracellular components. • This method distinguishes healthy cells with uncompromised membrane integrity (unlabeled) from non-healthy ones (colored).

×