Viability assay (WST)

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Need references of your contents Thyoon (talk) 08:28, 28 June 2016 (KST)

Introduction

Tetrazolium Reduction Assays[1]

Many tetrazolium compounds have been used access cellular viablity. The most commonly used compounds include: MTT, MTS, XTT, and WST-1. These compounds fall into two basic categories[1]: 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.

MTT Tetrazolium Assay Concept[1]

MTT.jpg

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was the first homogeneous cell viability assay developed for a 96-well format that was suitable for high throughput screening (HTS) (1). The MTT tetrazolium assay technology has been widely adopted and remains popular in academic labs as evidenced by thousands of published articles. The MTT substrate is prepared in a physiologically balanced solution, added to cells in culture, usually at a final concentration of 0.2 - 0.5mg/ml, and incubated for 1 to 4 hours. The quantity of formazan (presumably directly proportional to the number of viable cells) is measured by recording changes in absorbance at 570 nm using a plate reading spectrophotometer. A reference wavelength of 630 nm is sometimes used, but not necessary for most assay conditions. Viable cells with active metabolism convert MTT into a purple colored formazan product with an absorbance maximum near 570 nm (Figure 1). When cells die, they lose the ability to convert MTT into formazan, thus color formation serves as a useful and convenient marker of only the viable cells. The exact cellular mechanism of MTT reduction into formazan is not well understood, but likely involves reaction with NADH or similar reducing molecules that transfer electrons to MTT. Speculation in the early literature involving specific mitochondrial enzymes has led to the assumption mentioned in numerous publications that MTT is measuring mitochondrial activity. The formazan product of the MTT tetrazolium accumulates as an insoluble precipitate inside cells as well as being deposited near the cell surface and in the culture medium. The formazan must be solubilized prior to recording absorbance readings. A variety of methods have been used to

solubilize the formazan product, stabilize the color, avoid evaporation, and reduce interference by phenol red and other culture medium components (5-7). Various solubilization methods include using: acidified isopropanol, DMSO, dimethylformamide, SDS, and combinations of detergent. The amount of signal generated is dependent on several parameters including: the concentration of MTT, the length of the incubation period, the number of viable cells and their and metabolic activity. All of these parameters should be considered when optimizing the assay conditions to generate a sufficient amount of product that can be detected above background. The conversion of MTT to formazan by cells in culture is time dependent. Longer incubation time will result in accumulation of color and increased sensitivity up to a point; however, the incubation time is limited because of the cytotoxic nature of the detection reagents which utilize energy (reducing equivalents such as NADH) from the cell to generate a signal. For cell populations in log phase growth, the amount of formazan product is generally proportional to the number of metabolically active viable cells as demonstrated by the linearity of response in

Figure 2. Culture conditions that alter the metabolism of the cells will likely affect the rate of MTT reduction into formazan. For example, when adherent cells in culture approach confluence and growth becomes contact inhibited, metabolism may slow down and the amount MTT reduction per cell will be lower. That situation will lead to a loss of linearity between absorbance and cell number. Other adverse culture conditions such as altered pH or depletion of essential nutrients such as glucose may lead to a change in the ability of cells to reduce MTT. The MTT assay was developed as a non-radioactive alternative to tritiated thymidine incorporation into DNA for measuring cell proliferation. In many experimental situations, the MTT assay can directly substitute for the tritiated thymidine incorporation assay. However, it is worth noting that MTT reduction is a marker reflecting viable cell metabolism and not specifically cell proliferation. Tetrazolium reduction assays are often erroneously described as measuring cell proliferation without the use of proper controls to confirm effects on metabolism. Shortly after addition of MTT, the morphology of some cell types can be observed to change dramatically suggesting altered physiology. Toxicity of the MTT compound is likely related to the concentration added to cells. Optimizing the concentration may result in lower toxicity. Given the cytotoxic nature of MTT, the assay

method must be considered as an endpoint assay. A recent report speculated that formazan crystals contribute to harming cells by puncturing membranes during exocytosis (12). The observation of

extracellular formazan crystals many times the diameter of cells that grow longer over time make it seem unlikely that exocytosis of those large structures was involved. Growing crystals may suggest that marginally soluble formazan accumulates where seed crystals have begun to deposit. Reducing compounds are known to interfere with tetrazolium reduction assays. Chemicals such as ascorbic acid, or sulfhydryl-containing compounds including reduced glutathione, coenzyme A, and dithiothreitol, can reduce tetrazolium salts non-enzymatically and lead to increased absorbance values in assay wells (13-17). Culture medium at elevated pH or extended exposure of reagents to direct light also may cause an accelerated spontaneous reduction of tetrazolium salts and result in increased background absorbance values. Suspected chemical interference of test compounds can be confirmed by measuring absorbance values from control wells without cells incubated with culture medium containing MTT and various concentrations of the test compound.

Commercial Availability[1]

Commercial kits containing solutions of MTT and a solubilization reagent as well as MTT reagent powder are available from several vendors. For example:

•   CellTiter 96® Non-Radioactive Cell Proliferation Assay. Promega Corporation Cat.# G4000,

•   Cell Growth Determination Kit, MTT based. Sigma-Aldrich Cat.# CGD1-1KT, and

•   MTT Cell Growth Assay Kit. Millipore Cat.# CT02.

•   Thiazolyl Blue Tetrazolium Bromide (MTT Powder). Sigma-Aldrich Cat.# M2128.

The concentration of the MTT solution and the nature of the solubilization reagent differ among various vendors. The amount of formazan signal generated will depend on variety of parameters including the cell type, number of cells per well, culture medium, etc. Although the commercially available kits are broadly applicable to a large number of cell types and assay conditions, the concentration of the MTT and the type of solubilization solution may need to be adjusted for optimal performance.

Reagent Preparation[1]

MTT Solution

1.  Dissolve MTT in Dulbecco’s Phosphate Buffered Saline, pH=7.4 (DPBS) to 5 mg/ml.

2.  Filter-sterilize the MTT solution through a 0.2 µM filter into a sterile, light protected container.

3.  Store the MTT solution, protected from light, at 4°C for frequent use or at -20°C for long term storage.

Solubilization Solution[1]

1.  Choose appropriate solvent resistant container and work in a ventilated fume hood.

2.  Prepare 40% (vol/vol) dimethylformamide (DMF) in 2% (vol/vol) glacial acetic acid.

3.  Add 16% (wt/vol) sodium dodecyl sulfate (SDS) and dissolve.

4.  Adjust to pH = 4.7

5.  Store at room temperature to avoid precipitation of SDS. If a precipitate forms, warm to

37°C and mix to solubilize SDS.

MTT Assay Protocol[1]

1.  Prepare cells and test compounds in 96-well plates containing a final volume of 100 µl/well.

2.  Incubate for desired period of exposure.

3.  Add 10 µl MTT Solution per well to achieve a final concentration of 0.45 mg/ml.

4.  Incubate 1 to 4 hours at 37°C.

5.  Add 100 µl Solubilization solution to each well to dissolve formazan crystals.

6.  Mix to ensure complete solubilization.

7.  Record absorbance at 570 nm.

Resazurin Reduction Assay Concept[1]

Definition[1]

Resazurin is a cell permeable redox indicator that can be used to monitor viable cell number with protocols similar to those utilizing the tetrazolium compounds (26). Resazurin can be dissolved in physiological buffers (resulting in a deep blue colored solution) and added directly to cells in culture in a homogeneous format. Viable cells with active metabolism can reduce resazurin into the resorufin product which is pink and fluorescent (Figure 7). Addition of an intermediate electron acceptor is not required for cellular resazurin reduction to occur, but it may accelerate signal generation. The quantity of resorufin produced is proportional to the number of viable cells which can be quantified using a microplate fluorometer equipped with a 560 nm excitation / 590 nm emission filter set. Resorufin also can be quantified by measuring a change in absorbance; however, absorbance detection is not often used because it is far less sensitive than measuring fluorescence. The resazurin reduction assay is slightly more sensitive than tetrazolium reduction assays and there are numerous reports using the resazurin reduction assay in a miniaturized format for HTS applications (27). The incubation period required to generate an adequate fluorescent signal above background is usually 1to 4 hours and is dependent on the metabolic activity of the particular cell type, the cell density per well, and other assay conditions including the type of culture medium. The incubation period should be optimized and kept short enough to avoid reagent toxicity but long enough to provide adequate sensitivity. The major advantages of the resazurin reduction assay are that it is relatively inexpensive, it uses a homogeneous format, and it is more sensitive that tetrazolium assays. In addition, resazurin assays can be multiplexed with other methods such as measuring caspase activity to gather more information about the mechanism leading to cytotoxicity (28 and Figure 8). Multiplexing may require a sequential protocol to avoid color quenching by resazurin or direct chemical interference. For the multiplex example shown in Figure 8, resorufin fluorescence must be recorded first, followed by addition of the caspase reagent which ontains detergent to lyse cells and reducing compounds to convert remaining resazurin and reduce interference with collecting the second fluorescent signal. The disadvantages of the resazurin include the possibility of fluorescent interference from compounds being tested and the often overlooked direct toxic effects on the cells (Figure 9). Some protocols describe exposing cells to resazurin for several hours or even days; however, in some systems, changes in cell morphology can be observed after only a few hours of exposure suggesting interference with normal cell function (29). It is possible that exposure of cells to resazurin depletes reduced forms of nucleotides resulting in cytotoxic effects. Exposure of cells to resazurin is known to reduce the amount of ATP measured as a marker of cell viability. Figure 10 shows a decrease in ATP content of HepG2 cells exposed to resazurin for 4 and 24 hours.

Commercial Availability[1]

Commercial kits containing solutions of resazurin as well as resazurin powder are available from

several vendors. For example:

•   CellTiter-Blue ®Cell Viability Assay. Promega Corporation Cat.# G8081,

•   In Vitro Toxicology Assay Kit, Resazurin based. Sigma-Aldrich Cat.# TOX8-1KT,

•   alamarBlue ® —Rapid & Accurate Cell Health Indicator. Life Technologies, Inc. Cat.# DAL1100

•   alamarBlue® AbD Serotech Cat.# BUF012B

•   Resazurin sodium salt. Sigma-Aldrich Cat.# R7017-1G

Resazurin powder is readily available from chemical vendors; however, the resazurin dye content (% purity) and contamination with resorufin can lead to variability in assay results and the need to perform validation of each lot of reagent powder. Viability assay kits containing performance verified resazurin as the primary ingredient are available from different vendors; but the resazurin concentration and additional ingredients vary. The alamarBlue patent US 5,501,959 describes the use of poising agents to maintain the redox potential of the growth medium and prevent reduction of resazurin resulting in background signal (30). Preferred poising agents described include ferricyanide and ferrocyanide as well as methylene blue which can also serve as a redox indicator. The potential for undesired effects of additional ingredients in the proprietary alamarBlue formulation and the demonstrated performance equivalence of less complex formulations of highly purified resazurin in balanced saline solution should be considered when choosing an assay reagent.

Reagent Preparation[1]

1.  Dissolve high purity resazurin in DPBS (pH 7.4) to 0.15 mg/ml.

2.  Filter-sterilize the resazurin solution through a 0.2 μm filter into a sterile, light protected container.

3.  Store the resazurin solution protected from light at 4°C for frequent use or at -20°C for long term storage.

Resazurin Assay Protocol[1]

1.  Prepare cells and test compounds in opaque-walled 96-well plates containing a final volume of 100 µl/well. An optional set of wells can be prepared with medium only for background

subtraction and instrument gain adjustment.

2.  Incubate for desired period of exposure.

3.  Add 20 µl resazurin solution to each well.

4.  Incubate 1 to 4 hours at 37°C.

5.  Record fluorescence using a 560 nm excitation / 590 nm emission filter set.

A general disadvantage of both the tetrazolium and resazurin reduction assay protocols is the requirement to incubate the substrate with viable cells at 37°C for an adequate period of time to generate a signal. Incubation of the tetrazolium or resazurin reagents with viable cells increases the possibility of artifacts resulting from chemical interactions among the assay chemistry, the compounds being tested, and the biochemistry of the cell. Incubation also introduces an extra plate handling step that is not required for the ATP assay protocol described later. Extra plate manipulation steps increase the possibility of errors and are not desirable for automated assays for HTS.

WST assay

WST assay는 용액의 색변화를 통해 세포의 생존률을 측정하는 방법으로, 수용성 tetrazolium salt가 살아있는 세포와 반응하여 오렌지색의 수용성 formazan을 형성한다. MTT assay와 비교했을 떄, 형성하는 formazan이 수용성이기 때문에 녹이는 과정이 불필요하고 세포 독성이 매우 적어 같은 well에서 viability와 동시에 다른 실험들을 수행할 수 있다. MTT assay의 경우 세포 내 미토콘드리아의 활성을 측정하지만 WST assay의 경우 세포 내 모든 dehydrogenase와 반응하므로 세포의 상태를 보다 정확하게 반영하여 나타낼 수 있다.

장점

- soluble formazan을 형성하기 때문에 세포에 toxic한 유기용매를 처리하지 않아도 된다.

- MTT assay에 비해 높은 감도를 가지며, 측정 범위가 넓다.

Method

  1. 100 uL/well 씩 96 well에 세포 부유액을 넣고 배양시킨다.
  2. 세포의 population이 70-80%까지 자라면 상층액을 제거하고 well 당 최종 부피가 300 uL/well로 노출시키고자 하는 나노입자 stock을 세포에 일정시간 노출시킨다.
  3. 노출 후 나노입자에 의한 세포의 morphology 변화를 확인하기위해 microscopy를 이용하여 세포의 이미지를 측정한다.
  4. 상층액을 제거하고, well에 100uL/well 씩 WST solution을 넣은 후 incubator에서 1시간동안 배양시킨다.

5. Plate learder기의 사용 30분전에 켜놓고, 안정화시킨후 WST 측정에 해당하는 최대 파장인 450 nm로 설정한 후 흡광도를 측정한다.

References

  1. 1.00 1.01 1.02 1.03 1.04 1.05 1.06 1.07 1.08 1.09 1.10 1.11 Sittampalam GS, Coussens NP, Nelson H, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-. Available from: http://www.ncbi.nlm.nih.gov/books/NBK53196/