The overall goal of the following experiment is to use a fluorescence microplate assay to assess chemical effects on mast cell degranulation. In this protocol, the inhibitory effects of the anti-microbial agent triclosan is demonstrated without the use of an organic solvent. This is achieved by first plating mast cells into a 96 well plate to allow them to attach overnight and preparing triclosan in an aqueous solution without the use of organic solvents via heating.
The concentration of the Trian solution is then rapidly determined using ultraviolet visible spectroscopy. As a second step, cells are stimulated with IgE slash antigen or a calcium ionophore in the presence or absence of trian, which will cause the release of the mast cell degranulation marker beta hexosaminidase into the supernatant. The release of beta hexosaminidase into the cell supernatant is quantified using a beta hexosaminidase substrate whose fluorescent product is quantified in a microplate reader.
In order to measure the effect of triclosan or other test chemicals on mast cell degranulation results are obtained. That show triclosan inhibits antigen or calcium ion four activated degranulation in rat basophilic leukemia mast cells based on a fluorescent based microplate assay. The main advantage of this technique over earlier methods is that it is an inexpensive, rapid and highly sensitive alternative with the potential to lent itself to high throughput screening.
Additionally, the dissolution of trian into aqueous solution without the use some organic solvent avoids the confounding effects that many organic solvents have on mast cell function. This technique for triclosan concentration determination also has an advantage in that UV vs. Spectrometry is rapid and low cost.
This MA can provide insight into the effects of triclosan on mast cell degranulation, and this microplate as SA can also be applied for the screening of a wide variety of potential anti-allergy drug candidates and chemicals being tested for immunotoxic or endocrine disruptive effects. This protocol utilizes a rat basophilic leukemia mast cell line, or RB two H three, which is a widely accepted model of human mucosal mast cells or basophils. The RBL cells are cultured in a T 25 flask following standard procedures for trypsin and counting of cells.
A cell suspension with a density of 0.5 times 10 to the six cells per milliliter is made with RBL Media to plate cells dispense 100 microliters of cells per well in a flat black bottom 96 well plate. Following this plate template sheet to avoid systematic error, randomize how cells are added to wells. Mix the cell solution well, but gently between each set of three wells added to keep cells suspended during the plating procedure.
Once all the cells have been transferred to the 96 well plate, place the plate lid on the plate and incubate the plate in 5%CO2 at 37 degrees Celsius overnight. On the day of the degranulation assay, prepare Triclosan or TCS weigh out 0.0022 grams of TCS granules and transfer them to an erlenmeyer flask containing 250 milliliters of Ty Roads buffer and a stir bar and labeled as TCS buffer. In order to ensure all TCS has been transferred to the flask, use 10 milliliters from the measured 250 milliliters.
Ty Roads buffer to wash off the way boat. Add this buffer back to the TCS buffer flask. Place the TCS buffer flask onto a combination hot plate magnetic stir plate and set it to stir at a manageably high speed.
At the same time, place a control buffer erlenmeyer flask containing only 250 milliliters of Tyros buffer and no TCS onto a second combination hot plate magnetic stir plate and set it to stir at a similar speed. Heat the TCS buffer and control buffer solutions to 50 degrees Celsius while stirring constantly. Once the temperature has reached 50 degrees Celsius, set a timer for 90 minutes.
At the end of the 90 minutes, measure the concentration of the TCS solution using an ultraviolet visible spectrophotometer using the wavelength scan function on the spectrophotometer blank, the machine with one milliliter of the heated control buffer solution. Before scanning one milliliter of heated TCS buffer solution, record the absorbance value at 280 nanometers. To determine the concentration, use the Beer Lambert equation.
The TCS stock solution is typically between 20 and 30 micromolar begin preparations for the antigen stimulated degranulation assay. Once the TCS and control buffer solutions have been stirred at 50 degrees Celsius for 70 minutes as demonstrated, make up 0.1 micrograms per milliliter anti DNP mouse IgE in RBL media for sample wells to be sensitized, each well will require 100 microliters of IgE discard all media from the 96 well plate containing the RBL cells that was prepared the previous day and take the plate to the tissue culture hood randomly add 100 microliters of media IgE mixture to the 48 wells that should be stimulated. This mixture is not intended for spontaneous TX or background samples.
Next randomly add 100 microliters of plain RBL media only to TX spontaneous and background wells. Place the plate lid on the plate and incubate in 5%CO2 at 37 degrees Celsius for one hour. During the one hour incubation, prepare the exposure buffers first from the 249 milliliter sample of TCS buffer solution that has already been heated and stirred for 90 minutes.
Transfer 50 milliliters to a new 50 milliliter conical tube. Remove 20 microliters of this 50 milliliter aliquot and replace it with 20 microliters of a previously prepared one Microgram per milliliter D-N-P-B-S-A antigen. The final antigen concentration is 0.0004 micrograms per milliliter.
D-N-P-B-S-A vortex and invert the conical tube label. This tube one high TCS plus antigen plus BT.It will be used for dilution and highest TCS concentration exposure. Preparation of the other exposure buffers will not be shown in this video, but the procedures can be found in the accompanying text.
A total of four tubes will be prepared. Tube two contains antigen but no TCS and is used for TCS dilutions and zero micromolar TCS concentration. Exposure tube three contains high TCS but no antigen and is used for background tube four contains neither TCS nor antigen and is used for background and spontaneous samples.
The multiple concentrations of TCS to be tested in this assay should also be prepared at this time. For each dilution concentration, prepare a total volume of one milliliter in a sterile micro centrifuge tube. At the end of the one hour IgE incubation retrieve the 96 well plate from the incubator and discard all media into the sink in random fashion wash cells with BSA tyros buffer.
Using a combi tip release, 200 microliters of wash buffer onto the side of each well rather than directly onto the attached cells. In order to avoid disturbing the attached cells, repeat the wash a second time starting with the top section of the plate. Randomly add triplicates of 200 microliters each of the antigen solutions with correct concentrations of TCS to the corresponding wells.
Continue adding the appropriate solutions following this plate template. Once all appropriate solutions have been added to all wells, incubate the plate in 5%CO2 at 37 degrees Celsius for one hour. During the one hour incubation, prepare a new black bottom 96 well plate in a ice bucket and randomly add 100 microliters of cold, four mu flu genic substrate solution into the very bottom of each well.
At the end of the one hour incubation, retrieve the 96 well plate with the cells from the incubator and place it on ice in another ice bucket gently and without introducing bubbles, pipette the supernatant up and down four to five times going around the well for good mixing but not touching the cells adhered to the bottom of the plate while mixing. Systematically transfer a 25 microliter sample from each well to the new plate containing the flu genic substrate following the same ordering of samples as originally planned out. Pipette up and down to mix each sample thoroughly in the new well without introducing bubbles.
Incubate for 30 minutes at 37 degrees Celsius in 5%CO2 after 30 minutes. Randomly add 200 microliters of cold glycine carbonate buffer per well to fill each well to a total of 320 microliters. To avoid Triton X 100 spillover, add the buffer last to the Triton X 100 samples.
Subsequently run the plate in the fluorescence plate reader when heated to 50 degrees Celsius for 90 minutes. The ultraviolet visible absorbent spectrum for TCS produces a strong smooth curve between about 260 and 300 nanometers with a peak at 280 nanometers as indicated by the yellow line. In this figure.
Therefore, ultraviolet visible spectrometry is an important tool that can be utilized to calculate concentration. Since the published molar absorption coefficient at 280 nanometers is 4, 200 liters per mole per centimeter. In this example, the absorbance value at 280 nanometers is 0.11876, which indicates a TCS concentration of 28.28 micromolar.
The effect of TCS dissolved in aqueous buffer on mast cell degranulation was examined using a fluorescence based assay that records the level of beta hexosaminidase released from mast cells by detecting a fluoro substrate product. This figure shows results obtained for IgE sensitized RBL cells that were incubated for one hour in TCS buffer or control buffer and exposed to A-D-N-P-B-S-A antigen dose of 0.0004 micrograms per milliliter. This concentration of D-N-P-B-S-A elicited an average absolute degranulation response of 22.5%in the absence of TCS.
Statistically, significant inhibition of degranulation began at five micromolar where degranulation levels were 0.79 fold of the zero micromolar TCS control levels. As the TCS concentration increases, there is a greater dampening effect of TCS showing a strong dose response relationship. TCS at 20 micromolar almost completely abrogates the degranulation response to the levels roughly equivalent to spontaneous degranulation where no antigen is present.
The degranulation response of RBL mast cells stimulated with the calcium ion. A 2 3 180 7 in the presence of TCS was also investigated. A 2 3 1 8 7 was used as an alternative stimulant because it bypasses the FC epsilon RI crosslinking and other signaling events upstream of calcium influx, but still causes degranulation in the absence of TCS 180.
Ano molar of a 2 3 1 8 7 elicited an average absolute degranulation response of 25.1%Inhibition of degranulation was found with as little as one micromolar TCS as TCS concentration increases, so does the severity of the inhibition. At five micromolar, the degranulation response was 0.21 fold of the zero micromolar TCS control level. At 10 micromolar, it was 0.09 fold of the control level.
At 15 micromolar it was 0.077 fold of the control level, and at 20 micromolar the degranulation response was 0.09 fold of the control level. In fact, from TCS concentrations of five micromolar and higher levels of a 2 3 1 8 7 induced degranulation were found to be near the level of the spontaneous control where no a 2 3 1 8 7 is present taken together. The results from these experiments indicate that the molecular events targeted by TCS are likely downstream of calcium influx Following this procedure.
Other chemicals like those found in personal care products or in the environment can be tested in order to answer additional questions regarding their potential allergic effects In vitro. After watching this video, you should have a good understanding of how to prepare trian without the use of an organic solvent to rapidly check its concentration and to quantify the degranulation of RB two H three mast cells. Don't forget that working with particular test chemicals of interest can be extremely headless and precautious such as carefully following pertinent storage and handling information from the MSDS and from your institution.
Safety officers should always be taken while performing this procedure with a new test chemical.
