cell & molecular biology

Experiment 6

MTT Assay

Introduction:

Cell proliferation and death are essential processes for tissue generation and regeneration, organ development etc. in mammals and are usually under stringent control of extra and intracellular factors. Non-physiological alterations in levels of these factors lead to anomalous cytogenetic behavior of cells which in turn leads to cell transformation, uncontrolled cell growth - the initiating event for cancer development. Pharmaceutical research is hence largely focused on effects of drugs, cytotoxic agents and biologically active compounds which affect cytogenetics.

Multiple procedures are available for determination of cell proliferation and cytotoxicity. Simple and cheap methods for estimating cell viability (or death) are Trypan Blue exclusion and Erythrocin B staining. However, these methods are not sensitive enough and cannot be used for high throughput screening. Measuring the uptake of radioactive substances, usually tritium-labeled thymidine, is accurate but it is also time-consuming and involves handling of radioactive substances. Tetrazolium salts have been used to develop a quantitative colorimetric assay to estimate mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal 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 read on a multi-well scanning spectrophotometer or a standard ELISA reader and show a high degree of precision.

About the Assay

The MTT Cell Assay kit is designed for determination of cell viability and cell proliferation and/or effect of cytotoxic agent. This kit is based on the quantitative measurement of extracellular reduction of the yellow colored water soluble Tetrazolium dye 3-[4, 5- dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) to insoluble formazan crystals by metabolically active cells. This reduction is mediated by mitochondrial enzyme lactate dehydrogenase. When dissolved in a appropriate solvent, these formazan crystals exhibit purple color, the intensity of which is proportional to the number of viable cells and can be measured spectrophotometrically at 570nm. The assay procedure involves reconstitution of the pre-measured MTT reagent in the assay buffer, followed by its addition to the culture system. After dissolving the formazan crystals in the solubilization solution, results can be directly read on a suitable reader.

Applications:

• Cell proliferation: Quantification of changes in proliferative activity of cells caused by trophic factors, cytokines, and growth promoters

• Cell cytotoxicity: Evaluation of effects of inhibitors or inducers of apoptosis, cytotoxic reagents, carcinogens and toxins

• Drug discovery: High-throughput screening of various anti-cancer drugs

Materials

1. MTT reagent (powder)

2. Cell based assay buffer

3. Solubilization solution

Materials required but not provided in the kit:

a. Cells in appropriate medium with or without phenol red

b. Adjustable pipettes and a repeat pipettor

c. Flat-bottom 96-well microtiter plate for culturing the cells

d. 96-well plate reader capable of measuring the absorbance

Procedure

Preparation of MTT reagent:

Aseptically add 6ml of cell-based assay buffer in one MTT vial and completely dissolve the powder. MTT powder dissolves slowly in the buffer. Vigorous vortexing is needed to dissolve the powder completely. MTT solution should appear bright yellow in color.

(Note: For long term storage of the MTT reagent, it is recommended to filter sterilize using a 13mm, 0.22µ syringe filter. MTT reagent is light sensitive. Store the reconstituted reagent in amber colored bottle. If not consumed in single experiment, we recommend the storage of the reconstituted vial at -20^oC till further use.)

Preparation of cells:

Always use freshly harvested cells for assay. Seed the cells in a cell culture flask or dish in an amount appropriate for the assay and incubate at 37^oC in a 5% CO2 environment. Allow the cells to grow for up to 24 hours or till confluence is reached. Harvest the cells and use for assay.

(Note: The quantity of the cell suspension to be seeded in the medium depends upon doubling time of individual cell lines and seeding density to be used in the assay.)

Pre-assay optimization procedure:

Pre-assay optimization procedure needs to be performed once for each cell line to determine optimum plating density and incubation time.

1. Harvest the cells as explained in section 7.2.

2. Adjust the cell density to 1 x 10⁶ cells /ml.

3. Serially dilute the cell suspension from 1 x 10⁶ to 1 x 10³ cells/ml using appropriate culture medium.

4. Seed 100µl of each dilution in 96-well microtiter plate in triplicate.

5. Add medium control in triplicate.

6. Incubate the cells under appropriate conditions depending on the cell line under study.

7. Add 10µl of MTT to each well, including controls.

8. Wrap the plate with aluminium foil to avoid exposure to light.

9. Return the plate to the incubator for 2 to 4 hours.

10. Observe the cells at periodic intervals under an inverted microscope for presence of intracellular needle-shaped, dark purple coloured precipitate. Slow growing cell lines require longer time to develop formazan crystals.

11. When the purple precipitate is clearly visible under the microscope, add 100µl of solubilization solution to the wells.

12. Stir gently on a gyratory shaker for 30 minutes to enhance dissolution of the crystals. Mix the solution with the help of micropipette to allow crystals to dissolve. (Note: Plate can be incubated overnight after adding the solubilization solution followed by dissolution of crystals and reading.)

13. Read the absorbance on a spectrophotometer or an ELISA reader at 570nm with a reference wavelength higher than 600nm.

14. Determine the average values from triplicate readings at 570nm and subtract from this value the average value for blank (i.e. medium control) and average value at the reference wavelength.

Specific absorbance = Absorbance (570nm) (test) – Absorbance(570nm) (blank) –Absorbance(>600nm)(test).

15. Plot absorbance against cell density.

16. Number of cells to be used in the cell proliferation assay should lie within linear portion of the plot.

Assay procedures:

1. Seed 100µl of cell suspension in a 96-well microtiter plate at the required cell density, with or without the cell growth modifying agent.

(Note:

a) If the cell growth modifying agent is a cytokine, metabolite, growth factor or any other compound, add its required quantity in the culture system.

b) If the cell growth modifying agent is any kind of radiation or waves, treat the cells with them for required period of time.)

2. Incubate the plate at 37^oC in a 5% CO2 atmosphere for the required period of time.

3. After the incubation period, remove the plates from incubator and add MTT reagent to a final concentration of 10% of total volume. This volume should be same as the volume used while determining optimum cell density.

4. Wrap the plate with aluminium foil to avoid exposure to light.

5. Return the plates to the incubator and incubate for 2 to 4 hours.

Note: Incubation time varies for different cell lines. Incubation time should be kept constant while making comparisons. Some cell lines may require for up to 24 hours.

6. Remove the plate from incubator after incubation.

7. After incubation period, add 100µl of solubilization solution to each well.

8. Stir gently on a gyratory shaker for 30 minutes to enhance dissolution of the crystals. Mix the solution with the help of micropipette to allow crystals to dissolve.

Note: Plate can be incubated overnight after adding the solubilization solution followed by dissolution of crystals and reading.

9. Read the absorbance on a spectrophotometer or an ELISA reader at 570nm with a reference wavelength of 630nm.

10. Subtract the average 570nm absorbance values of the control wells from the average 570nm absorbance values of corresponding experimental wells.

11. Measure the absorbance of all the assay wells again at a wavelength 630nm. Subtract these values from the values obtained at 570nm. This reading will help you eliminate non-specific readings from your assay result.

Plot the absorbance values on the Y-axis and your experimental parameters on the X-axis.

Interpretation of Data

1. The linear portion of the MTT curve depicts maximum sensitivity to changes induced by experimental parameters.

2. Test values higher than control values indicate increase in cell proliferation and viability and vice versa.

TRYPAN BLUE Assay

Introduction:

Cell proliferation and death are essential processes for tissue generation and regeneration, organ development etc. in mammals and are usually under stringent control of extra and intracellular factors. Non-physiological alterations in levels of these factors lead to anomalous cytogenetic behavior of cells which in turn leads to cell transformation, uncontrolled cell growth - the initiating event for cancer development. Pharmaceutical research is hence largely focused on effects of drugs, cytotoxic agents and biologically active compounds which affect cytogenetics.

Multiple procedures are available for determination of cell proliferation and cytotoxicity. Simple and cheap methods for estimating cell viability (or death) are Trypan Blue exclusion and Erythrocin B staining. However, these methods are not sensitive enough and cannot be used for high throughput screening. Measuring the uptake of radioactive substances, usually tritium-labeled thymidine, is accurate but it is also time-consuming and involves handling of radioactive substances. Quantitative measurement of reduction of Resazurin dyes is an alternate simple, safe and accurate method for determination of cell proliferation. This assay is similar to tetrazolium assay, except that it has the optional advantages of using fluorescent detection methods and omission the of solubilization step.

About the Assay

The EZBlue^TM Cell Assay Kit is based on reduction of the water soluble, redox dye EZBlue^TM. The dye itself is dark blue in color and has intrinsic fluorescence. The fluorescence changes when EZBlue^TM is reduced by metabolically active cells to the pink resorufin product. The spectral properties of resorufin allows the molecule to be detected using either absorbance or fluorescence. However, fluorescence is the preferred method of evaluating the end-point of the reaction as it is more sensitive than absorbance. Dead or non-viable cells are incapable of this reduction and do not contribute to the fluorescence generated by viable cells. The specific mechanism responsible for the reduction of EZBlue^TM is unknown, but probably involves the same cellular processes which generate equivalents such as NADH. In comparison to other methods of evaluating cell viability, which require tedious steps like reagent transfer, washing, crystal dissolution, repeated aspiration etc., the use of EZBlue^TM is a single step process of addition of the reagent to cells. This ensures a rapid assay with minimal errors and facilitates its use in large scale hi-throughput screenings.

Applications

• Cell proliferation: Quantification of changes in proliferative activity of cells caused by trophic factors, cytokines, radiations and growth promoters

• Cell cytotoxicity: Evaluation of effects of inhibitors or inducers of apoptosis, cytotoxic reagents, carcinogens and toxins

• Drug discovery: High-throughput screening of various anti-cancer drugs

Materials:

• EZBlueTM Solution

• Cells in appropriate medium

• Adjustable pipettes and a repeat pipettor

• 96-well plate for culturing the cells: 96-well plate reader or spectrophotometer capable of measuring the absorbance at 580nm and >600nm or fluorescence at 560nmEX/590nmEM

Procedure:

Preparation of reagent: Bring the reagent to room temperature before use.

Preparation of cells:

Always use freshly harvested cells for assay. Seed the cells in a cell culture flask or dish in an amount appropriate for the assay and incubate at 37^oC in a 5% CO₂ environment. Allow the cells to grow for up to 24 hours or till confluence is reached. Harvest the cells and use for assay.

(Note: The quantity of the cell suspension to be seeded in the medium depends upon doubling time of individual cell lines and seeding density to be used in the assay.)

Procedure for determination of optimum cell density and incubation period to be used in the assay:

Use the procedure given below to determine optimum plating density and incubation period for cell line. This is a preliminary experiment that should be performed before staring the actual assay.

1. Harvest the cells as explained above.

2. Seed 100µl cells in 96-well plate at various cell densities, above and below the cell density expected to be used for the assay.

3. Add 10µl EZBlue^TM reagent. (10µl equals to 10% of the total culture volume per well).

4. Wrap the plate in aluminium foil and incubate at 37^oC in a 5% CO₂ environment.

5. Read the absorbance at 580nm keeping >600nm as a reference wavelength, or fluorescence a 560nmEX/590nmEM at each hour following plating for first 6 - 8 hours.

6. Process the data and calculate percentage reduction of EZBlue^TM by using formulae mentioned above.

Assay procedures

Procedure for determining cell proliferation:

1. Seed 100µl cell suspension in a 96-well plate at the required density. Treat them with cell growth modifying agent.

(Note: If the cell growth modifying agent is a cytokine, metabolite, growth factor or any other compound, add its required quantity in the culture system. If the cell growth modifying agent is any kind of radiation or waves, treat the cells with them for required period of time.)

2. Incubate the plate at 37^oC in a 5% CO₂ atmosphere for required period of time.

3. After the incubation period, remove the plates from incubator and add EZBlue^TM reagent to a final concentration of 10% of total volume. This volume should be same as the volume used while determining optimum cell density. Wrap the plate with aluminium foil to avoid exposure to light.

4. Return the plates to the incubator and incubate for 1 to 8 hours.

(Note: Incubation time varies for different cell lines. Incubation time should be kept constant while making comparisons.)

5. Remove plate from the incubator after incubation, followed by mixing on a gyratory shaker.

6. Read the absorbance at 580nm keeping >600nm as a reference wavelength, or fluorescence at 560nmEX/590nmEM.

7. The plate can be reread at a later time in culture period as long as cells remain viable.

(Note: This time depends on the properties of the cell line as well as nature of the compound under test).

Procedure for determining cell cytotoxicity:

1. For adherent cells: Seed 100µl cell suspension in a 96-well plate at required cell density, without the test agent. Allow the cells to adhere to the culture plate for about 24 hours. Add the desirable quantity of the test agent for suspension cells: Seed 100µl cell suspension in a 96-well plate at required cell density. Add the test agent immediately in the desired quantity

2. Incubate the plate for desired period at 37^oC in a 5% CO₂ atmosphere.

3. After the incubation period, remove the plates from incubator and add 10µl EZBlue^TM reagent. (10µl equals to 10% of the total culture volume per well.)

4. Wrap the plate with aluminium foil to avoid exposure to light.

5. Return the plates to the incubator and incubate for 1 to 8 hours.

(Note: Incubation time varies for different cell lines. Incubation time should be kept constant while making comparisons.)

6. Remove plate from the incubator after incubation, followed by mixing on a gyratory shaker.

Reference Materials

Trypan blue assay: https://www.youtube.com/watch?v=dSxH9xNmhWg

MTT assay:https://www.youtube.com/watch?v=vn6enA6lSKs

Questions

1. How many cells are required to obtain an efficient reading with the MTT cell proliferation assay?

2. MTT reagent stores at what degree?

3. What will be the result of drug when it has no effect in cells?

4. why do we use MTT assay?

5. What dose trypan blue bind to?

6. How dose trypan blue distingusih between live and dead cells?