This method can help answer key questions about photodynamic therapy, otherwise known as PDT. The questions that can be answered by using this method include the duration of incubation of a photosensitizer, or the use of different parameters in the experiment such as changing the temperature by heating or cooling the cells. This technique can help facilitate the in vitro development of novel photosensitizers, protocols, and indications for PDT.
Begin by plating two times 10 to the fourth cells in two milliliters of culture medium into each well of a six well plate using sterile technique for a 24 hour incubation at 37 degrees Celsius and 5%carbon dioxide. The next day, wash the cells with at least two milliliters of PBS and treat the cultures with zero, zero point five, one, or two millimeter dilutions of freshly prepared 5-ALA. Then place the plate on a 36-37 degree Celsius heating block for 30 minutes protected from light.
During the procedure, we recommend during the incubation phase that researchers use a heating block to achieve a consistent temperature throughout the incubation period. Additionally, we recommend covering the cells during the incubation period with aluminum foil to prevent earl activation of the photosensitizer. At the end of the incubation, wash the cells in each well with two milliliters of PBS.
And refresh the cultures with two milliliters of fresh culture medium for blue light irradiation. Before irradiating the cells, warm up the blue light device at a 417 nanometer output wavelength for one 1, 000 second cycle and use a photometer to measure the irradiance at the cell surface, adjusting the distance of the light to achieve the appropriate irradiance according to the intensity of the light source. Then place the 5-ALA treated cells onto a black surface under the blue light for 1, 000 seconds for a total fluence of 10 joules per square centimeter.
During photo activation, we recommend placing the cells on a black surface to prevent light reflection and inconsistent dosing. At the end of the phototherapy treatment, wash the cells in each well with two milliliters of PBS and detach the cells with one milliliter of 0.25%trypsin-EDTA per well. After three to five minutes, confirm detachment and deactivate the trypsin with 10%FBS and PBS.
Transfer the cells from each well into individual five milliliter flow tubes and collect the cells by centrifugation. Add 200 microliters of flow buffer with conjugated annexin-V antibody to each sample. Re-suspend the cells and place the tubes into the incubator for 20 minutes to allow for annexin-V binding.
At the end of the incubation, add three microliters of 7-AAD to each sample and incubate again at room temperature for five minutes followed by a sample analysis done according to standard flow cytometric analysis protocols. After five ALA and blue light phototherapy treatment, a dose-dependent increase in cell apoptosis is observed. To calculate the percentage of cells undergoing apoptosis, the percentage of cells in the annexin-V high, 7-AAD low and annexin-V high, 7-AAD high quadrants are added.
Inconsistencies in the five ALA incubation period length, incubation temperature, or photo activation irradiation dose may alter the phototherapy efficacy, resulting in a temperature-dependent increase in apoptosis, for example. Following this procedure, other techniques including RTQPCR and western blot may be performed to determine how PDT alters gene expression and protein activity. This method allows researchers who are interested in photodynamic therapy to discover the effects on skin cancer cells and other skin diseases in vitro.
