The overall goal of this coculture protocol is to measure the effects of ionizing radiation on Caco-2 epithelial cell monolayer permeability and tight junction function in the presence or absence of peripheral blood mononuclear cells. This method can help answer key question in the radiation biology and radioimmunotherapy fields about possible synergistic effects between ionizing radiation exposure and immune cell functions. The main advantages of this technique are that different stimuli and cell population can be tested and observed phenomena uncoupled through ad hoc complementary measurements.
The radiation procedures aims at considering the Caco-2 cell monolayer as tumor volume is irradiated with the appropriate beam and accurate dosimetry as is case of radiotherapy in patient. One week before their irradiation seed five times 10 to the fifth Caco-2 cells in two milliliters of complete medium into one sterile one micrometer pore diameter cell culture insert per well in a six well cell culture plate. Add three milliliters of fresh complete medium into the bottom compartment of each well and place the cells in a humidified cell culture incubator at 37 degrees Celsius and 5%Co2 for seven days.
On the last day of the incubation add 25 milliliters of Ficoll to a 50 milliliter conical tube. And carefully layer 25 milliliters of freshly collected whole blood over the Ficoll. Separate the cells by density gradient centrifugation.
And use a Pasteur pipette to transfer the peripheral blood mononuclear cells or PBMC at the interface into a new 15 milliliter conical tube. Then wash the isolated PBMC in two 10 milliliter PBS washes. And culture the PBMC in fresh complete medium for no more than three to five hours in the cell culture incubator.
Set the photon x-ray energy to a six megavolt peak. And place the Caco-2 cell culture on a 1.4 centimeter thick plexiglass sheet within the trajectory of the x-rays and 100 centimeters from the source of radiation. Then place a 0.57 centimeter thick bolus on each sample to guarantee equilibrium of the back-scattered radiation component and the charged particles.
And use a flat and symmetric 20 by 20 square centimeter radiation field and a dose rate of three grays per minute to irradiate the cells. To assess Caco-2 cell metabolic activity and viability, seed two times 10 to the fifth Caco-2 cells in each well of a 24-well plate 24 hours before their irradiation in 1.25 milliliters of complete medium. Then return the cells to the cell culture incubator for 21 hours.
The next day add 100 microliters of five milligrams per milliliter MTT solution to each well and incubate the cells for another three hours. After washing the cells with one milliliter of PBS, add 500 microliters of dimethyl sulfoxide to each well to dissolve any formazan crystals released by the Caco-2 cells and evaluate the absorbance with a multi-well plate reader at a lambda of 570 nanometers. To assess Caco-2 cell viability, wash the cells with one milliliter of PBS per well followed by detachment of the cells with 100 microliters of Trypsin-EDTA solution for two minutes at 37 degrees Celsius and 5%CO2.
Stop the reaction with 500 microliters of complete medium and transfer the cells to individual 1.5 milliliter microcentrifuge tubes for centrifugation. Resuspend the pellets in 50 microliters of PBS per tube and mix the resulting cell suspensions with an equal volume of Trypan Blue viability dye solution. After three minutes at room temperature, count the number of viable unstained and nonviable stained cells in the hemocytometer.
To assess the transepithelial electrical resistance or TEER of the Caco-2 cells, immediately after irradiation transfer half of the Caco-2 cell culture inserts into a new plate with three milliliters of fresh complete medium in each well and half of the insert cultures into a new plate seeded with two times 10 to the sixth PBMC per three milliliters of complete medium per well. Then place a TEER chopstick electrode into the cell culture insert every hour for the first six hours and then every three hours until 48 hours after irradiation. For Western blot analysis, 48 hours after their irradiation lyse Caco-2 cells and PBMC with 40 microliters per one times 10 to the sixth cells of cell lysis buffer.
When lysing and scraping the Caco-2 cells, take care not to detach the porous membrane from the insert, which can result in a loss of cell lysate and a biased result. Store the lysed cell samples at minus 20 degrees Celsius. After quantifying the total protein amount in each cell lysis sample by the bicinchoninic acid method, add equal volumes of Laemmli sample buffer supplemented with beta-mercaptoethanol to each total protein sample in individual microcentrifuge tubes.
Heat the samples at 95 degrees Celsius for five minutes. Then collect the denatured proteins by centrifugation and load equal total protein volumes from each sample in a four to 20%precast gel. Run the samples for one hour at 120 volts.
Then use a semi-dry electric transfer system to transfer the proteins onto a polyvinylidene fluoride membrane. Next place the membrane in a container and block the nonspecific binding sites with 5%nonfat dry milk in PBS supplemented with 0.2%Tween 20 for 60 minutes at room temperature with gentle agitation. At the end of the blocking incubation, wash the membrane three times with 10 milliliters of 0.2%PBS Tween 20 for five minutes per wash and label the membrane with the appropriate primary antibodies of interest for one hour at room temperature with gentle agitation.
After overnight incubation at four degrees Celsius with gentle agitation, wash the membrane three times with 0.2%PBS Tween 20 as just demonstrated and incubate the membrane with the appropriate horseradish peroxidase conjugated secondary antibodies for 60 minutes at room temperature with gentle agitation. After three PBS washes, incubate the membrane with an enhanced chemiluminescent solution kit. Then image the resulting film with an appropriate scanning system and quantify the results with a suitable image analysis program.
Irradiation up to 10 grays does not alter Caco-2 cell metabolic activity at 24 or 48 hours post irradiation, although the cell viability decreases over time at both doses. TEER assessment of noncocultured Caco-2 cells after exposure to two grays of irradiation reveals consistent TEER values up to 48 hours after irradiation. After 10 grays of irradiation, however, the cells demonstrate a prolonged decrease in TEER beginning at three hours post-irradiation.
Coculture with PBMCs results in a reduction of TEER that is evident from three hours post-irradiation at both doses until 30 hours post-irradiation, at which point the TEER values appear to remain relatively consistent. Western blot analysis of tight junction proteins reveals no differences in Claudin-1 or Occludin expression in response to irradiation and/or PBMC coculture, while large fluctuations are observed in various scaffold proteins. Further, NF-kappa B total protein is not affected at either dose of irradiation, whereas XIAP protein levels are upregulated four-fold at both doses.
Using this procedure other biological stimuli like Enterobacteria can be added to answer additional questions about how different biological stimuli can modify the response of the good monolayer to ionizing radiation exposure. After watching this video you should have a good understanding of how to measure the impact of ionizing radiation and immune cells on Caco-2 cell permeability and tight junction complex expression.
