Cell viability and proliferation assays

This ppt contains, tests for the cell viability and proliferation.

1. CELL VIABILITY AND PROLIFERATION ASSAYS Presented by:- Mohit Agrawal M.Pharm (Pharmacology) D e l h i P h a r m a c e u tical S c i e nces a n d R e s e arch U n i v e r s ity , N e w D e l h i

2. INTRODUCTION Cell viability assay is a homogeneous method to determine the no. of viable cell in culture. Cell viability assay method is used to estimate the number of viable cells in multi-well plates. Cell-based assays are often used for screening collections of compounds to determine if the test molecules have effects on cell proliferation or show direct cytotoxic effects that eventually lead to cell death.

3. DNA SYNTHESIS PROLIFERATION ASSAYS BrdU Cell Proliferation Assay Cell proliferation may be studied by monitoring the incorporation of a radioisotope, [3H]-thymidine, into cellular DNA, followed by autoradiography. Alternatively, 5-bromo-2′-deoxy-uridine ( BrdU assay ) may be used instead of thymidine. Cells that have incorporated BrdU into their DNA are easily detected using a monoclonal antibody against BrdU and an enzyme- or fluorochrome-conjugated secondary antibody.

4. EdU Proliferation Assays  Base click EdU proliferation assays provide an efficient method for fluorescence detection of replicating DNA. The modified nucleoside EdU is added to live cells and is incorporated into replicating DNA. Cu-induced click chemistry allows rapid attachment of fluorescent probes to the EdU. This provides for a quantitative way to monitor cells that are proliferating.

5. METABOLIC PROLIFERATION ASSAYS Assays that measure metabolic activity are suitable for analyzing proliferation, viability, and cytotoxicity. The reduction of tetrazolium salts such as  MTT ,  XTT , and  WST-1  to colored formazan compounds or the bio-reduction of resazurin occurs only in metabolically active cells. Actively proliferating cells increase their metabolic activity, while cells exposed to toxins will have decreased activity.

6. MTT CELL PROLIFERATION ASSAYS MTT(3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide; thiazolyl blue) is a water soluble tetrazolium salt yielding a yellowish solution when prepared in media or salt solutions lacking phenol red. Dissolved MTT is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by dehydrogenase enzymes. This water-insoluble formazan can be solubilized using isopropanol or other solvents, and the dissolved material is measured spectrophotometrically using absorbance as a function of concentration of converted dye.

7. XTT CELL PROLIFERATION ASSAYS In contrast to MTT, the cleavage product of XTT is soluble in water. The tetrazolium salt XTT is cleaved to formazan by a complex cellular mechanism. This bio-reduction occurs in viable cells only, and is related to NAD(P)H production through glycolysis. The amount of formazan dye formed directly correlates to the number of metabolically active cells in the culture.

8. WST-1 CELL PROLIFERATION ASSAYS The stable tetrazolium salt WST-1 is cleaved to a soluble formazan by a complex cellular mechanism that occurs primarily at the cell surface. This bio-reduction is largely dependent on the glycolytic production of NAD(P)H in viable cells. The amount of formazan dye formed directly correlates to the number of metabolically active cells in the culture.

9. LUMINISCENT CELL PROLIFERATION ASSAYS Because ATP is an indicator of metabolically active cells, the number of viable cells can be assessed based on the amount of ATP available. The ATP viability cell luciferase offers a highly sensitive homogenous assay for quantifying ATP in cell cultures. This kit employs firefly luciferase to oxidize D-Luciferin and the resulting production of light to assess the amount of ATP available in cell cultures. By relating the amount of ATP to the number of viable cells, the assay has wide applications, ranging from the determination of viable cell numbers to cell proliferation to cell cytotoxicity.

10. FLOURESCENT DYE PROLIFERATION ASSAY CFSE Labeling 5(6)- Carboxyfluorescein diacetate N- succinimidyl ester (CFSE)  is a popular choice for measuring the number of divisions undergone by a cellular population. Upon entering the cell, CFSE is cleaved by intracellular esterase to form the fluorescent compound and the succinimidyl ester group covalently reacts with primary amines on intracellular proteins. Upon division, the fluorescence intensity of each daughter cell is halved enabling simple detection of the number of cell divisions by flow cytometry.

11. Live/Dead Cell Double Staining Live/Dead Cell Double Staining  can be utilized for simultaneous fluorescence detection of viable and dead cells. Calcein-AM  is a highly lipophilic and cell membrane-permeable dye. Though Calcein-AM itself is not a fluorescent molecule, the Calcein generated from Calcein-AM by esterase in a viable cell emits a strong green fluorescence (λex 490 nm, λem 515 nm). Calcein-AM therefore only stains viable cells.  In contrast, the nuclei-staining dye  Propidium Iodine  cannot pass through a viable cell membrane. It reaches the nucleus by passing through disordered areas of dead cell membrane, and intercalates with the DNA double helix to emit red fluorescence (λex 535 nm, λem 617 nm). Since both Calcein and PI-DNA can be excited with 490 nm light, simultaneous monitoring of viable and dead cells is possible with a single-excitation fluorescence microscope.

12. 3D CELL CULTURE LIVE/DEAD/TOTAL CELL TRIPLE STRAINING The  Cell Viability Imaging Kit  is a three-color assay that can be used with 2D and 3D cell cultures for simultaneous fluorescence staining of viable cells ( Calcein -AM), dead cells (Propidium Iodide/PI), as well as total cells (Hoechst 33342). Calcein-AM  fluoresces green on binding calcium, relying on esterase activity present only in metabolically-active viable cells. Propidium Iodide  (PI) is a nuclear dye that is excluded by the membrane of live cells, but passes through the damaged membrane of dead cells, intercalating with the DNA to emit a strong red fluorescence. Hoechst 33342  is a DNA staining dye that exhibits low cytotoxicity. It fluoresces blue and is used as an indicator of total cell count.

13. TRYPAN BLUE CELL COUNTING Trypan B lue is one of several stains recommended for use in dye exclusion procedures for viable cell counting. This method is based on the principle that live (viable) cells do not take up the blue dye, whereas dead (non-viable) cells do. Cell viability can be calculated using the ratio of total live/total cells (live and dead). Staining also facilitates the visualization of overall cell morphology. Trypan Blue has a greater affinity for serum proteins than for cellular protein.

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