The overall goal of this procedure is to assess the antifungal activity of the primary human immune cells in real time using fluorescent aspergillus reporter conidia and live cell video microscopy. This method can help to answer key questions regarding the immune responses against fungal infections in specific patient populations and particularly the innate immune response occurring early upon exposure to fungi. The main advantage of this technique is that it can be used to identify specific defects in innate and antifungal immunity such as phagocytosis cell migration killing an inhibition of fungal growth.
To begin, streak aspergillus fumigatus strain AF293 carrying the fluorescent dsRed reporter gene over agar prepared in two T75 flasks. Incubate the cultures at 37 degrees Celsius, 5%CO2 for seven days to obtain approximately 10 to the 9th conidia. After the incubation, immerse the cultures in 20 milliliters of PBS with 0.05%Tween 80 and collect A fumigatus conidia.
Then using a 40 micrometer cell strainer, filter the resulting suspensions into a 50 milliliter tube to remove hyphal fragments. Once the suspensions are filtered, centrifuge them at 805 times G for 10 minutes. Then discard supernatants and resuspend the pellets in 20 milliliters of sterile PBS.
Finally, pool the samples and centrifuge the resulting suspension one more time. Resuspend the pellet in five milliliters of PBS and transfer one milliliter aliquots of the obtained conidial suspension into microcentrifuge tubes. Centrifuge the aliquots at 9, 300 times G at room temperature for 10 minutes.
Then carefully aspirate the supernatant and resuspend the conidial pellet in one milliliter of 0.05 molar sodium bicarbonate with a PH of 8.3. To label the conidia, add 10 microliters of the biotin stock solution to the microcentrifuge tube, cover the sample with aluminum foil and incubate for two hours on a rocker at four degrees celsius. After centrifuging the biotin labeled sample for 10 minutes at 9, 300 times G, carefully remove the supernatant.
Then resuspend the conidial pellet in one milliliter of a 100 micromolar Tris-HCL solution with a PH of 8.0. And incubate this suspension at four degrees celsius for one hour with gentle agitation to deactivate the unbounded biotin. Centrifuge the sample at 9, 300 times G for 10 minutes.
Carefully remove the resulting supernatant, wash the pellet with one milliliter of sterile PBS and resuspend the pellet in one milliliter of PBS. Then add 10 microliters of a Streptavidin-AF633 stock solution to one milliliter of the conidial suspension. Incubate this suspension covered with aluminum foil for 40 minutes at room temperature on a rocker.
Centrifuge the sample at 9, 300 times G for 10 minutes. Then carefully remove the supernatant and resuspend the conidial pellet in one milliliter of PBS. Using a hemocytometer, count the conidia at a one to 1, 000 dilution and then adjust the conidial concentration to 3.6 times 10 to the 6th conidia per milliliter with CO2 independent medium.
Finally, wrap the suspension in foil and store at four degrees celsius. Collect and dilute a sample of human blood with PBS. Then use a syringe with an iron needle to underlay the blood sample with 15 milliliters of a medium for isolation of mononuclear cells.
Centrifuge the sample at 630 times G for 20 minutes with no break and low acceleration. Collect with a Pasteur pipette the center PBMC layer localized between the yellow serum and the transparent lymphocyte isolation solution layer. Then transfer the collected suspension into a new 50 milliliter tube.
Dilute the PBMC with PBS to obtain 50 milliliters of the suspension and wash the cells. After washing, mix 20 microliters of the cell suspension with 160 microliters of PBS and 20 microliters of trypan blue and count the cells with a hemocytometer. When counted, dilute to an appropriate concentration in a 15 milliliter tube, centrifuge the cells for 10 minutes at 515 times G, and then resuspend them using 40 microliters of a buffer solution per one times 10 to the 7th of cells.
Add 10 microliters of CD14 MicroBeads per one times 10 to the 7th cells and mix well. Incubate the sample at four degrees celsius for 15 minutes, making sure to mix the tube every five minutes. After conjugation, wash the cells in one milliliter of the buffer solution per one times 10 to the 7th cells and centrifuge the suspension at 515 times G for 10 minutes.
Then resuspend up to one times 10 to the 8th cells in 500 microliters of the buffer solution. Mount one column that will be used with a single sample on a magnetic separator following the manufacturer's instructions and wash the column once with 500 microliters of buffer solution. Load 500 microliters of the cell suspension onto the column.
Once it is dry, wash the column thrice using buffer solution. Place a new 15 milliliter tube underneath the column and detach the column from the magnetic separator. Then using one milliliter of buffer solution, elute the cells from the column into the tube.
Wash and resuspend the eluted cells in RPMI medium. After adjusting the cell density to six times 10 to the 5th cells per milliliter, seed 200 microliters of the suspension on a 35 millimeter glass based imaging dish. Incubate the culture overnight at 37 degrees celsius with 5%CO2.
To isolate neutrophils, remove all serum and most of the lymphocyte isolation solution from the blood sample remaining after the PBMC harvest. Make sure not to remove the thin white band on top of the red pellet. Keeping the cells on ice, next, lyse the red blood cells and centrifuge the remaining neutrophils at 394 times G.Then wash the cells twice with cold PBS and then resuspend in one milliliter of a CO2 independent medium.
For a flow cytometry, adjust the cell density to six times 10 to the 5th cells per milliliter and add 400 microliters of the cell suspension to a 24 well plate. Add 200 microliters of the same cell suspension to a 35 millimeter glass based imaging dish for microscopic examination. To visualize dsRed and Alexa Fluor 633 during microscopy, preheat a microscope heater and turn on the microscope and the computer.
Next, add 100 microliters of three times 10 to the 6th per milliliter FLARE conidial suspension to the appropriate wells of the imaging dish. Record the time that FLARE conidia are added. Mount the imaging dish on the microscope stage, then in the acquisition settings, set the laser power to 10%and exposure time to one second.
And adjust the camera sensitivity for a clear but not overexposed image of dsRed and AF633 and set up a list of stage points. Initialize imaging when all points are in focus, the fluorescent channels are optimized, and the cycle time and duration are set. Presented here are images taken during a simultaneous culture of neutrophils and FLARE conidia.
Green fluorescent signal corresponding to the AF633 staining is visible in both live and dead conidia. While dsRed fluorescence can be observed exclusively in live conidia. Thus among all the detected conidia, only those simultaneously stained with both dyes and visible as magenta spots are live.
Phagocytosis and killing of A fumigatus conidia co-cultured with mouse alveolar macrophage cells was quantified using flow cytometry. In the analysis, the bystander macrophages are marked with an orange gate while macrophages associated with dead conidia and positive only for AF633 staining are shown within the blue gate. In addition, the dead conidia do not reveal calcofluor white staining, indicating their intracellular localization.
Live conidia stained with both dsRed and AF633 are marked with a red gate, these conidia are also internalized within macrophage cells as evidenced by the diminished calcofluor white staining. Finally, the influence of A fumigatus on the migration of human neutrophils was assessed, revealing increased movement and velocity of the immune cells in response to swollen conidia compared to resting conidia. Once mastered, this technique can be done in eight hours for a single cell population.
While performing this procedure, it is important to remember to keep your cells on ice. Following this procedure, other methods like the measurement of reactive oxygen species or cytokinesis can be performed to provide further information regarding the innate antifungal immune response in the same cell population. After it's development, this technique paves the way for researchers in the field of fungal immunology to explore phagocytosis and antifungal killing using fungal pathogens in primary immune cells.
After watching this video, you should have a good understanding of how to assess innate antifungal immune responses of primary human immune cells in real time using fluorescent aspergillus reporter conidia and live cell video microscopy. Don't forget that working with human blood can be hazardous and appropriate PPE precautions should always be taken to prevent the transmission of infection.
