The overall goal of this procedure is to quantify pathogenic yeasts in association with host cells without plating colony forming units. First, infect the macrophage cells with H capsulation or C albicans yeast cells stain the infected macrophage cells with aine orange and propidium iodide to highlight viable yeast cells. Then using an imaging cytometer capture a series of images and analyze the size and shape parameters to accurately enumerate the yeast cells.
Results from image cytometry can effectively be used to quantify pathogenic yeasts in association with host cells. I first had the idea for this method when doing research in the field of fungal pathogenesis, in which I had to repeatedly plate and count colony forming units for quantification of the pathogenic fungus Histoplasma capsule. The main advantage of image cytometry over the standard method of CFU enumeration is its increased speed as it eliminates the labor and time intensive steps of plating, incubating and counting multiple serial dilutions of CFU When comparing growth of wild type and mutant strains within host cells.
This method eliminates the potential bias introduced by differing abilities to initiate quality growth on solid media. Generally, individuals new to this method might be challenged by the image cytometry. Instrumentation, and software.
Begin the procedure by culturing the macrophages at desired density. In 24 well plates add the fungal cells in log phase growth, then incubate for 90 minutes to allow for phagocytosis. Next, wash the macrophages three times with PBS to remove the extracellular fungi incubate for the experimentally defined time points prior to sample analysis at low multiplicity of infection, the infected macrophage cells remain viable for several days, and samples can be analyzed approximately every 12 to 24 hours.
After three washes with PBS, add 0.5 milliliters of sterile water to the infected macrophage cells. Incubate for five minutes at room temperature to liberate the fungi from the macrophage cells. Then harvest the lysate to a sterile tube and keep on ice.
Transfer 20 microliters of the lysate to a separate tube. Add 20 microliters aine orange propidium iodide solution for immediate image cytometric analysis. Perform tenfold dilution of the lysates in media.
Then plate 100 microliters of each dilution in duplicate. On HMM aros plates. Incubate the plates in a humidified chamber at 37 degrees Celsius with 5%carbon dioxide for seven to eight days.
Manually count colonies on the plates, displaying a minimum of 100 and a maximum of 1000 distinct colonies. To collect the live macrophages, wash the cells three times with PBS. Then add 0.5 milliliters of PBS and incubate for 30 minutes at four degrees Celsius.
Remove the macrophages from tissue culture wells by gentle pipetting. Transfer the liquid to a sterile tube and store on ice. To set up the accelerometer instrument, insert the fluorescence optics modules into the system and make sure they're locked in place.
Turn on the image cytometer in the assay dropdown menu of the software. Select the preset assay type and cell type for viability. The assay is optimized for aine orange and propidium iodide detection for detection of candida albicans infection.
The assay is optimized for GFP detection. Now open the options tab and click on take background image After the operation has completed, click on preview brightfield image for the A OPI viability measurement. Mix 20 microliters of sample with 20 microliters of A OPI and pipette into accounting chamber for candida albicans infection rate.
Mix the sample well and pipette it directly into counting chamber. After the cells have settled in the chamber, insert the counting chamber into the image cytometer for image acquisition. Bring the sample into focus, then click on count.
When the image acquisition operation is complete, remove the disposable counting chamber and dispose of it properly. For A OPI measurement, the concentration and viability results are displayed on the counting results page to determine the candida albicans infection rate. After obtaining the counting results, click on export.
Then send the data to FCS express four for cell population analysis of GFP. In FCS express, import the data file and plot the results in a fluorescence histogram. Next, apply a cell population gate to the histogram to determine the population percentages of candida albicans infected cells.
This experiment monitors growth of macrophage cells infected by hcaps Lotum yeast at defined time points. Samples are stained and analyzed by image-based cytometry. The liberated yeast cells from laced macrophages are identified by the Soter vision software.
Here, the absolute number of live yeasts detected is greater than yeast capable of colony formation on solid medium. These data highlight the presence of significant numbers of viable, but non cultivatable cells. These bone marrow derived macrophages were infected with C albican yeast cells expressing green fluorescent protein under the control of the A DH one promoter.
Multiplicities of infection ranging from 0.1 to 10 resulted in incremental increases in GFP intensity within infected macrophages, as well as total percentage of infected macrophages Once mastered. This technique can be done in only minutes per sample if it's performed properly. While attempting this procedure, it's important to remember to set the parameters such that only fungal cells not host cells are counted.
After watching this video, you should have a good understanding of how to use image cytometry to quantify pathogenic yeasts in association with host cells. Don't forget that working with pathogenic fungi can be extremely hazardous, so wear personal protective equipment and follow proper biohazard waste disposal procedures.
