This method can help answer key questions in the pulmonary care field, such as why do patients who survive hospital acquired pneumonia have such poor long term outcomes? The advantage of this method is that it's simple and reliable, which means that anybody who comes in the lab can be taught it and the next day they're generating useful and repeatable data. Though these analytical methods can be applied to provide insight into the infection of cultured cells in vitro, they can also be applied to animal models and human patients.
To produce cytotoxic supernatant, wash a 150 centimeter dish of endothelial cells with HBSS, and dilute a P.aeruginosa colony to an absorbance of 0.25 at 540 nanometers. Further dilute the bacterial cells`to allow a multiplicity of infection of 20 to one in 20 milliliters of HBSS and seed the bacteria onto the plate of endothelial cells. Then place the infected cells in a 37 degrees Celsius and 5%CO2 incubator for four to five hours.
It is essential that the incubation of the bacteria and the endothelial cells occur for an appropriate length of time. If the incubation is too short, the amyloids will not be released into the supernatant. If the incubation is too long, the cells will actually lyse and release all of their contents into the supernatant.
The indicator that we use for an appropriate duration of incubation is the formation of gaps in the endothelial monolayer. When gaps can be observed in the cell monolayer by light microscopy harvest the supernatant for centrifugation to remove any cellular debris. Decant the supernatant into a syringe that is equipped with a 0.2 micron filter and pass the supernatant through the filter to remove any contaminating bacteria.
Then set aside 1.5 milliliters of sterile supernatant for cytotoxicity testing and freeze the rest of the sample at minus 80 degrees Celsius. To assess the cytotoxicity of the harvested supernatant, add the 1.5 milliliter aliquot of filter sterilized supernatant to a single well of a six well plate containing a confluent pulmonary microvascular endothelial cell culture. Place the plate in a CO2 incubator for 21 to 24 hours.
Then obtain images of regions of the treated and control cultures. Import the images into a custom imagej macro and adjust the contrast to 15%saturated pixels. Duplicate the contrast adjusted images and use subtract background to obtain high contrast images of both the intact cells and the gap area within the field of view.
Subtract this high contrast image from the original image and use the image calculator and function to combine the resulting image with the original image. Use the threshold function to convert the combined image to a mask with the gaps in black and the intact cells in white. And use the binary erode function to remove any noise from the image.
Then use the area fraction to measure the ratio of black to white pixels within the resultant image. And plot and express the fractional areas for each treatment time point, as the percent of the maximal gap area. To quantify the amyloids within the supernatant, add 20 microliters of freshly prepared and filtered 50X thioflavin T stock solution to one milliliter of PBS in a one milliliter spectrophotometer cuvette, and load the diluted sample onto a spectrofluorometer.
Measure the baseline fluorescence emission using a 425 nanometer excitation. Scanning the fluorescence emission from 450 to 575 nanometers in two nanometer increments. Then set the instrument to perform a time lapse scan using a 425 nanometer excitation and 482 nanometer emission with data acquisition every 0.2 seconds for 60 seconds.
Pause the scan after 20 seconds and 10 microliters of filter sterilized supernatant to the cuvette. After mixing by inversion, reload the cuvette onto the spectrofluorometer and resume the time based scan for the final 40 seconds. Upon completion of the time lapse, perform a final fluorescence emissions spectrum scan using the original scan settings.
The addition P.aeruginosa to confluent layers of pulmonary microvascular endothelial cells induces gap formation between the cells. Using imagej to assess supernatant cytotoxicity as demonstrated during the first 12 hours of treatment, still healthy confluent cell monolayers can be visualized as all white regions within the microscopic field. However, by 18 hours after the addition of supernatant collected from PA 103 infected cultures, gaps in the monolayer can be detected with the area of the culture dish devoid of cells increasing in a linear manner until 36 hours of treatment, at which point almost no intact cells can be observed.
Similar results can be obtained using lactate dehydrogenase release as a marker of cytotoxicity. With lactate dehydrogenase first detected at 18 hours after the addition of the cytotoxic supernatant and increasing in a linear fashion until maximal cell killing is measured at 36 hours. The supernatants can also be assessed by immunoblot analysis or by measuring the change in thioflavin T fluorescence intensity due to confirmational changes induced by amyloid binding to determine the presence of cytotoxic amyloids in the supernatants.
After watching this video you should have a good understanding how to generate cytotoxic supernatants following the infection of endothelial cells with citomonsis aeruginosa. In addition, you should be well versed in the types of assays needed to character and analyze the cytotoxins that are present in those supernatants.
