EZcount MTT Cell Assay Kit 1 - HiMedia Labs

1 EZcountTM MTT Cell Assay Kit Product Code: CCK003. 1. Introduction dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide Cell proliferation and death are essential processes for (MTT) to insoluble formazan crystals by metabolically tissue generation and regeneration, organ development active cells. This reduction is mediated by mitochondrial etc. in mammals and are usually under stringent control of enzyme lactate dehydrogenase. When dissolved in a extra and intracellular factors. Non-physiological appropriate solvent, these formazan crystals exhibit purple alterations in levels of these factors lead to anomalous color, the intensity of which is proportional to the number cytogenetic behavior of cells which in turn leads to cell of viable cells and can be measured transformation, uncontrolled cell growth - the initiating spectrophotometrically at 570nm. event for cancer development. Pharmaceutical research is hence largely focused on effects of drugs, cytotoxic The Assay procedure involves reconstitution of the pre- agents and biologically active compounds which affect measured MTT reagent in the Assay buffer, followed by its cytogenetics.

2 Addition to the culture system. After dissolving the formazan crystals in the solubilization solution, results can Multiple procedures are available for determination of cell be directly read on a suitable reader. proliferation and cytotoxicity. Simple and cheap methods for estimating cell viability (or death) are Trypan Blue exclusion and Erythrocin B staining. However, these 3. Applications methods are not sensitive enough and cannot be used for Cell proliferation: Quantification of changes in high throughput screening. Measuring the uptake of proliferative activity of cells caused by trophic radioactive substances, usually tritium-labeled thymidine, factors, cytokines, and growth promoters is accurate but it is also time-consuming and involves Cell cytotoxicity: Evaluation of effects of inhibitors handling of radioactive substances. Tetrazolium salts have or inducers of apoptosis, cytotoxic reagents, been used to develop a quantitative colorimetric Assay to carcinogens and toxins estimate mammalian cell survival and proliferation.

3 The Drug discovery: High-throughput screening of Assay detects living, but not dead cells and the signal various anti-cancer drugs generated is dependent on the metabolic state of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be 4. Kit contents read on a multi-well scanning spectrophotometer or a Contents Kit Code standard ELISA reader and show a high degree of CCK003- CCK003- Storage Code Description precision. 1000* 2500**. MTT reagent 30mg x 2 30mg x 5. TC247 4oC. (powder) vials vials 2. About the Assay Cell based . TL1111 1 x 20ml 2 x 20ml RT**. TM Assay buffer The EZcount MTT Cell Assay kit is designed for Solubilization . determination of cell viability and cell proliferation and/or TCL094 1 x 125ml 2 x 125ml RT**. solution effect of cytotoxic agent. This kit is based on the *. Sufficient for 10 microplates (1000 assays). quantitative measurement of extracellular reduction of the **.

4 Sufficient for 25 microplates (2500 assays). yellow colored water soluble Tetrazolium dye 3-[4, 5- **. RT- Room Temperature . Quantities supplied in excess to compensate operational losses 1. 5. Materials required but not provided in the Incubation period kit Different cell lines may have different properties such as metabolic activity and doubling time and hence Cells in appropriate medium without phenol red respond to MTT differently. For this reason, plating Adjustable pipettes and a repeat pipettor density and incubation period for every cell line Flat-bottom 96-well microtiter plate for culturing the should be optimized to obtain results in linear range. cells Formation of formazan crystals can be checked by 96-well plate reader capable of measuring the observing the cells under inverted microscope absorbance periodically during incubation. The crystals appear as needle-shaped and dark purple coloured intracellular 6.]

5 General guidelines precipitates. Longer incubation period may be It is important to optimize experimental factors like cell required if adequate amount of crystals are not density, incubation time, media composition and formed. concentration of the agents under investigation prior to Read the plates within 1 hour of addition of use of EZcountTM MTT Cell Assay Kit. Procedure for solubilization solution. optimizing cell density is outlined in section Culture Medium Assay controls Phenol red interferes with the measurement of Include appropriate Assay controls formazan; therefore the cell culture media used for 1. Medium control (medium without cells) this Assay should not contain phenol red. 2. Cell control (medium with cells but without the High protein content in the culture medium may lead experimental drug/ compound) to precipitation on addition of solubilization solution. 3. Vehicle control (medium containing the Serum is the major factor contributing to high protein experimental drug or compound but no cells) content of culture medium.

6 Maximum acceptable Extracellular reducing components such as ascorbic concentration of fetal bovine serum is 10%. However, acid, cholesterol, alpha-tocopherol, dithiothreitol sera with higher protein content than FBS should be present in the culture media may reduce the MTT to used at lower concentrations. formazan. To account for this reduction, it is important to use the same medium in control as well Temperature as test wells. Temperature affects the performance of the Assay because of its effect on enzymatic rates. It is crucial Accuracy to run the Assay at a uniform temperature to ensure To obtain statistically significant data, perform the reproducibility across a single plate or among stacks Assay in triplicates or more. of several plates. Since absorbance or fluorescence Accuracy of the Assay depends on pipetting skills of readings measured at room temperature, it is the personnel. Inappropriate addition and mixing important to ensure adequate equilibration of Assay practices may result in erroneous and false-positive or plates after removal from a 37oC incubator to avoid false-negative results.

7 Differential temperature gradients. Stacking large Use of a repeating pipettor is recommended to pipette numbers of Assay plates in close proximity should be reagents. This saves time and helps maintain more avoided to ensure complete temperature equilibration. precise incubation times. Measurement of absorbance Pipette tip should be equilibrated with the reagent before use. This is carried out by slowly filling up the Absorbance can be read with a filter in the tip with reagent and gently expelling the contents wavelength range of 550-600nm (primary several times. wavelength). Care should be taken so that no bubbles are Reference wavelength (for non-specific readings). introduced into the wells during pipetting or mixing should be higher than 650nm. of the reagents. 2. 7. Directions for use 10. Observe the cells at periodic intervals under an inverted microscope for presence of intracellular Users are advised to review entire procedure before needle-shaped, dark purple coloured precipitate.

8 Starting the Assay Slow growing cell lines require longer time to develop formazan crystals. 11. When the purple precipitate is clearly visible Preparation of MTT reagent under the microscope, add 100 l of solubilization Aseptically add 6ml of cell based Assay buffer in one solution to the wells. MTT vial and completely dissolve the powder. MTT. 12. Stir gently on a gyratory shaker to enhance powder dissolves slowly in the buffer. Vigorous dissolution of the crystals. vortexing is needed to dissolve the powder 13. Read the absorbance on a spectrophotometer or an completely. Concentration of the resulting solution is ELISA reader at 570nm with a reference 5mg/ml. MTT solution should appear bright yellow in wavelength higher than 650nm. color. 14. Determine the average values from triplicate (Note: readings at 570nm and subtract from this value the For long term storage of the MTT reagent, it is average value for blank ( medium control) and recommended to filter sterilize using a 13mm.)

9 Average value at the reference wavelength. syringe filter. MTT reagent is light sensitive. Store the reconstituted Specific absorbance = Absorbance (570nm) (test) . reagent in amber colored bottle. If not consumed in Absorbance(570nm) (blank) Absorbance(>650nm). single experiment, we recommend the storage of the (test). reconstituted vial at -20oC till further use.). 15. Plot absorbance against cell density. Preparation of cells 16. Number of cells to be used in the cell proliferation Always use freshly harvested cells for Assay . Seed the Assay should lie within linear portion of the plot. cells in a cell culture flask or dish in an amount appropriate for the Assay and incubate at 37oC in a Assay procedures 5% CO2 environment. Allow the cells to grow for up 1. Seed 100 l of cell suspension in a 96-well to 24 hours or till confluence is reached. Harvest the microtiter plate at the required cell density, with cells and use for Assay .

10 Or without the cell growth modifying agent. (Note: The quantity of the cell suspension to be (Note: seeded in the medium depends upon doubling time of a) If the cell growth modifying agent is a individual cell lines and seeding density to be used in cytokine, metabolite, growth factor or any the Assay .). other compound, add its required quantity in the culture system. Pre- Assay optimization procedure b) If the cell growth modifying agent is any kind Pre- Assay optimization procedure needs to be of radiation or waves, treat the cells with performed once for each cell line to determine them for required period of time.). optimum plating density and incubation time. 2. Incubate the plate at 37oC in a 5% CO2. atmosphere for the required period of time. 1. Harvest the cells as explained in section 3. After the incubation period, remove the plates 2. Adjust the cell density to 1 x 106 cells /ml. from incubator and add MTT reagent to a final 3.