The overall aim of this procedure is to perform a live cell video microscopy experiment to study phagocytosis of the human fungal pathogen candida albicans. This is accomplished by first culture in candida albicans overnight, so they enter their stationary phase. The next step is to plate J 7 74 macrophages in 35 millimeter glass bottomed imaging dishes and culture them overnight.
On the next day, candida albicans are stained with pH sensitive Zi and the acidic organelles of the macrophages are stained using liso tracker red. The final step is to make a video of the Phagocytosis assay where the J 7 7 4 macrophages are cultured with the candida albicans. Ultimately, the resulting video can show stage Pacific analysis of macrophage migration recognition, engulfment and FGA zone maturation.
The main advantage of this technique over existing methods, such as immuno cyto chemistry, is that live video microscopy can be used to break down, observed differences into individual stages, such as migration, engulfment, and phagosome maturation, which may be affected. Differentially, Though this method can provide insight into the phagocytosis of Canada Acan. It can also be applied to other systems such as other pathogens, phagocytes, including dendritic cells and neutrophils.
This culture requires advanced preparation of SC minus U agar plates. They can be stored refrigerated, begin by streaking candida albican serotype, a strain CI four plus CLP 10 from glycerol stocks stored at minus 80 degrees Celsius onto an SC U agar plate. Incubate the plate at 30 degrees Celsius until colonies form typically 48 hours.
At this point, the plate can be stored at four degrees Celsius until ready to use. Now pick a single colony and culture it in five milliliters, SCU or medium overnight at 30 degrees Celsius on a rocker set at 200 RPM. This will generate stationary phase candida albicans.
Add 10 microliters of the candida albicans overnight culture to 990 microliters of PBS at pH 7.4 and perform a cell count using a hemo cytometer to visualize the candida albicans during the phagocytosis assays stain 100 million candida using Zi for 10 minutes at room temperature in the dark. Next, remove the unbound fitzy by washing the candida albicans three times in PBS to wash centrifuge the candida at 3000 times G for five minutes. Remove the sup natant and re suspend the pellet in one milliliter of PBS Reese.
Suspend the final pellet in PBS at a concentration of 1 million cells per microliter. For this protocol, have prepared a culture of J 7 7 4 0.1 macrophages in 75 centimeter square tissue culture flasks in supplemented DMEM at 37 degrees Celsius with 5%carbon dioxide from the flask. Scrap the macrophages and transfer them to a 50 milliliter Falcon tube Centrif.
Use the macrophages at 600 times G for five minutes to obtain a cell palate. Remove the supinate and resuspend the pellet in 10 milliliters of pre-war supplemented DM dmm. Now count the cells using a hemo cytometer and plate 1 million cells in a 35 millimeter glass based imaging dish with two milliliters of supplemented DMM medium.
Incubate the dish overnight at 37 degrees Celsius and with 5%carbon dioxide the next day. Prior to imaging, replace the supplemented DMM medium with two milliliters of prewarm carbon dioxide independent medium containing one micromolar of Liso tracker red DND 99, and the same supplements as the dmm. The microscope setup needs to include an inverted stage and environmental chamber heated to 37 degrees Celsius and excitation emission filters for the chosen stains being fitzy and trixy.
In this example, an applied precision delta vision core is utilized. First turn on the microscope heater so that the control chamber is warm to 37 degrees Celsius. Then turn on the microscope and computer and load the imaging software, mount the imaging dish on the microscope stage and adjust the focus to find the J 7 74 0.1 macrophages.
Optimize the appearance of trixi and DIC images by adjusting the percentage of transmitted light and exposure times. Remove the imaging dish and add 3 million fitsy stained candida albicans to the dish. Record the time and return the dish to the stage, optimize the appearance of fitzy images if necessary, and set up the points of interest in the imaging software if required.
Now commence the imaging when all points are in focus and the channels are optimized. Capture fitzy, trixi and DIC images every minute for six hours. This is the most critical stage in the procedure.
Time has to be taken to set up the microscope correctly. If the movies are under or overexposed or if they're outta focus, they'll be unusable and the experiment will need to be repeated. This representative video shows candida albicans uptake by the murine macrophage cell line J 7 74 0.1.
During a six hour live cell video microscopy experiment, the candida albicans in green was stained using the pH sensitive dye fitsi, which was quenched during acidification of the macrophage phagosome. Thus confirming when the candida were internalized, the acidic compartments of the macrophages were stained with a red fluorescent dye. Notice the variation in the number of candida albicans ingested by individual J 7 74 0.1 macrophages in this experiment, 82%of J 7 74 0.1 macrophages engulfed at least one candida albican cell within the six hours.
The mean number of candida albicans taken up per macrophage was 3.4, but interestingly, macrophages could ingest up to 16 fungals cells. The data allows observation of macrophages and candida albicans prior to and during recognition and uptake. Candida albicans hyer can continue to grow within the macrophage, which can result in macrophage lysis.
After watching this video, you should have a good understanding of how to perform live cell video microscopy experiments using candida albicans on the J 7 7 4 0.1 microphage like cell line. You should be able to set up overnight cultures of both cell types and perform a phagocytosis assay, which will enable stage specific analysis of migration recognition and engulfment, and also phagosome maturation.
