Introduction MTT Assay – Cytotoxicity of ResveratrolIntroductionCell culture techniques are used for many experiments in modern cell biology laboratories. Phenomena can often be studied in a more controllable environment than with intact organisms or isolated tissues. The number and viability of cells being used in an experiment are important parameters that may affect the outcome as should be kept as consistent between experiments as possible. Several methods exist for cell enumeration and for quantifying the cell viability of a culture. The dye exclusion assay and the MTT assay are simple methods for quantifying these parameters and will be discussed in more detail below. A third type of te st (seeding and plating efficiencies) involves plating a known number of cells and then examining them over time to see how many are able to attach to the surface and undergo cell division to form colonies. This is a more difficult and time-consuming assay, requiring a great deal of practice than the first two assays. For a dye exclusion assay, a dye such as trypan blue is used. Substances like trypan blue are not transported into healthy cells and cannot penetrate the cell membrane if it is structurally intact. When cells become damaged (and/or die) the membranes often become compromised in their ability to exclude chemicals such as trypan blue, and the dyes leak into the cells and they become stained. If a population of cells is stained with trypan blue, then the total number of cells (whether stained or not) in the population as well as the viability of the cells (the percentage of cells that are “alive” and unstained) can be determined based on the criterion of membrane integrity. Unstained cells represent healthy, living cells, and stained cells have compromised membranes and may either be dead or may not be able to recover from the damage they have sustained and will die in the future. The MTT assay is the one we will use in this experiment to obtain a relative number of cells between cultures. The MTT assay uses the compound 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). This compound can be metabolized by the mitochondrial enzyme, succinate dehydrogenase to form blue-colored, water-insoluble crystals. This reaction requires that a mitochondrial membrane potential exists across the mitochondrial inner membrane and, therefore is a measure of cell viability based on the criterion of the cell possessing functional mitochondria (and thus, functional oxidative phosphorylation). If the mitochondrial membrane potential is reduced or eliminated, ATP synthesis will also be reduced or cease completely. Most cells cannot survive long with impaired ATP synthesis or may already be dead.The MTT assay can be used for many different types of experiments including measuring cell proliferation (the increase in the size of the cell population due to cell division over time), and measuring cytotoxicity (the killing of cells within a population). If multiple cell populations are incubated with the MTT compound for a uniform time period, then the amount of color produced by the metabolism of the MTT will be directly proportional to the number of cells in the culture. We will be using the MTT assay to test the cytotoxic effects of the substance resveratrol which has been identified as one of the main ingredients in red wine that has anticancer properties and is believed give red wine its health benefits. If this ingredient is to truly be a health benefit it would have to inhibit proliferation or kill cancer cells without having adverse effects on normal cells. We will te st 2 different cell lines in this experiment. The design of this experiment is quite simple. We will seed cells from a single population in the 24 wells of a multiwall plate so that the number of cells placed in each well should be approximately the same. Some cells will remain untreated (as controls) to provide data for how the “normal” cell proliferation rate is for this population. Then other cells will be treated with different concentrations of resveratrol. We will te st the cells after 24 hours to see how many live cells (based on the presence of functional mitochondria) are present. Each plate will contain the cells of one cell line, but we will be testing the effects of the te st substance on two different cell lines, one normal cell line and one cancer cell line.The normal cell line we will be using a normal human fibroblast cell line. The cell line we will te st is the FA cells; fibroblasts from a patient with Friedreich’s ataxia. The cell line behaves very much like a normal human fibroblast line. The second cell line we will te st is a human basophilic leukemia cell line (RBL-2H3). It is a cancerous cell line. In this assay, the cells will be seeded at a moderate cell density. Each well will contain 0.5 ml of Eagle’s minimal essential medium (EMEM) containing glucose (4.5 g/L), glutamine (2 mM) and which has been supplemented with sodium pyruvate (1 mM), sodium bicarbonate (1.5 g/L), and fetal bovine serum (10%). All incubations of these cells are at 37°C and under an atmosphere of 5% CO2, to maintain a neutral pH.Pre-lab set up: The cells were seeded in the multi-well plates on Day 0. Then after 48-72 hours the cells were treated with the various concentrations of resveratrol. A total of 12 plates were prepared and treated for each of the two cell types. Each student will receive a plate containing one cell type and we will later collect the data from all the replicates to analyze as one experiment. Each cell line will be tested 6 times (n=6). Cell Set up: Cells from each cell line were seeded in the 24 wells of a multi-well plate with 0.5ml of medium in each well. The cells were incubated for 48-72 hours and then were treated with the resveratrol at 7 different concentrations (plus a set of wells which were not treated with resveratrol as control samples). Serial Dilution: (A total of 8 different concentrations of resveratrol were prepared through a serial dilution from a stock solution that was 20mg dissolved in 4ml of dimethylsulfoxide (DMSO). – Aliquots of 0.5ml DMSO were placed in each of 6 microcentrifuge tubes. – An aliquot (0.5ml) of the stock resveratrol was added to the first tube of DMSO and it was mixed well. – The other 6 concentrations of resveratrol were prepared by similar dilutions. – An additional tube contained only DMSO (called the vehicle) as a control for cells treated with no resveratrol.(Calculate the stock concentrations of the resveratrol that were used. The first stock solution was prepared by dissolving 20mg of resveratrol in 4ml of DMSO. This is equal to 5.00mg/ml (see Tube A). The serial dilution at each step was to take 0.5ml of the resveratrol and add 0.5ml of DMSO (i.e. a 1 plus 1 dilution or 1 to 2 dilution). (Remember when calculating a dilution, use the equation C1V1 = C2V2 where C1 equals the concentration in the starting solution, V1 equals the volume of the first solution used to make the dilution, V2 equals the volume of the second solution, and C2 is the concentration in the diluted sample (the unknown in the equation.) Tube A = 5.00mg/ml Tube B = _______Tube C = _______Tube D = _______Tube E = _______Tube F = _______Tube G = _______Tube H = 0mg/ml (this tube has DMSO with no resveratrol) Protocol for MTT Assay: A. Treatment of cells with resveratrol: (This part of the experiment was done on the day before the MTT assay will be carried out.) 1. Aliquots of 5 μl of the stock concentration of resveratrol were added to each of 3 wells of the plate. 2. Aliquots of 5 μl of each dilution was added to 3 wells each for the other 6 dilutions of the resveratrol. 3. The 8th set of 3 wells were treated with 5 μl of DMSO only as the control samples (no substance condition). The plates were incubated at 37°C until the lab meets on Monday.
(NOTE: The dilutions that were prepared will be diluted 1:100 when added to the wells of the plate. You will need to express the concentrations of resveratrol based on the values in the wells, rather than the concentrations in the “stock” tubes.)Stock ConcentrationConcentration of resveratrol in each well A. __________________________B. __________________________C. __________________________D. __________________________E. __________________________F. __________________________G. __________________________H. __________________________(NOTE: All materials from these plates and treatments below will be collected in collection containers on your benches.)B. Treatment with MTT:1. Aspirate the medium containing the substance (or DMSO) from all wells using a disposable Pasteur pipet (DPTP). Be careful to minimally disturb the cells in the well. If you are rough the cells will be scraped off the surface. 2. Replace with 0.50 ml of fresh medium (see above) containing 0.5 mg/ml MTTand incubate the plate for approximately 30-60 minutes at 37oC. (Use a serological pipet for this step.)C. Quantification of metabolized MTT:1. Carefully aspirate the medium containing MTT from each well with a Pasteur pipet and dispose of it in the 50ml conical tube waste container. 2. Rinse the wells with two (2) volumes of 0.5 ml of PBS. Add 1.0 ml of isopropanol to each well and dissolve the Formazan crystals.
Transfer the samples to 1ml cuvettes using a disposable Pasteur pipet and measure the absorbance (Abs570nm) for each sample.
D. Data analysis – Raw Data and Summarized Data(NOTE: Remember to use the concentrations of resveratrol of substance that were in the wells (i.e. the concentration of resveratrol that the cells were exposed to in the experiment. Do not use the stock concentrations.) Part 1. Raw Data (Table 1) – Due Thursday 4-22-21 (by 5:00pm)Calculate the average absorbance values for the triplicates for each concentration of the substance. Do this for both cell types.
Calculate the standard deviation for the triplicates for each concentration of the resveratrol. Do this for both cell types.
Create a data table for the raw data (Table 1). with the absorbance values, the averages, and the standard deviation values for both cell types.
Part 2. Summarized Data (Table 2) – Due Thursday 4-22-21 (by 5:00pm)Use the control (DMSO only) wells to calculate relative absorbance values.
For each resveratrol condition, divide the average absorbance values by the average absorbance value for the control (DMSO containing wells) and multiply by 100 to determine the relative absorbance.
Repeat this calculation for each of the other concentration conditions and for both cell types.
Part 3. Graph for Raw Data (Figure 1) – DRAW THIS GRAPH ON GRAPH PAPER BY HAND. – Due Tuesday 4-27-21 (by 5pm) Plot the data for absorbance values as a function of the concentration of resveratrol in the wells of the multi-well plate. Use only the average absorbances with the standard deviation (mean + 1 S.D.). Plot the data for both cell types on one graph.
Part 4. Graph for Summarized Data (Figure 2) – DO THIS GRAPH ON THE COMPUTER (using the averages only) with the data for both cell types on one graph.- Due Tuesday 4-27-21 (by 5pm)Plot the data for the relative absorbances (y-axis) as a function of the resveratrol concentration (x-axis) and determine the mathematical relationship between these variables.
Part 5. Determinaton of the LD50 – Due Tuesday 4-27-21 (by 5pm)For each cell line that you can, calculate the concentration of resveratrol that caused 50% cell death. This value is the lethal dose for 50% of the cells.
Can you calculate this value for each cell type over the range of resveratrol concentrations tested? What do the values tell you about the ability of this te st substance to kill normal and cancer cells? (i.e. What can you conclude about the ability to selectively kill cancer cells relative to normal cells?)
Remember that you need to limit your conclusion(s) to the cells lines that were tested. Do not generalize to all cells.
