To treat human-derived macrophages with Trichoderma stromaticum conidia using sterile tweezers, add a round glass cover slip to each well of a 24 well plate. Count the PBMC suspension isolated from human blood and adjust the volume to plate eight times 10 to fifth cells per well to a final volume of one milliliter with R10 medium. After plating, use an inverted microscope.
Observe the cell morphology. Incubate the cells at 37 degrees Celsius in 5%carbon dioxide for seven days, replacing the medium with fresh R10 medium every two days for the differentiation of the monocytes into macrophages. After seven days, observe the cell morphology using an inverted microscope.
Then remove the medium from each well and wash the cells with one milliliter of PBS to remove debris and non-adherent cells. Next, prepare T.stromaticum conidia suspension at a final concentration of eight times 10 to the fourth conidia per milliliter in R10 medium. Add one milliliter of suspension to each well to obtain a multiplicity of infection of one to 10.
Challenge the macrophages for three hours at 37 degrees Celsius under 5%carbon dioxide. After incubation, remove the medium and wash the cells with PBS to remove the nonphagocetized conidia. Then add one milliliter of R10 medium to each well and incubate the plate at different times.
After the corresponding incubation, remove the medium and add one milliliter of PBS to that well. Then using tweezers and a needle, remove the round glass cover slips from the wells. Next, stain the cover slips using the components of the staining kit.
Once the cover slips are dry, fix them on a glass slide using a mounting agent. For conidial viability assay, challenge the macrophages with T.stromaticum conidia and incubate the plate for different times at 37 degrees Celsius under 5%carbon dioxide. After each incubation, wash the cells twice with PBS to remove the unphagocytosed conidia.
Then add 0.5 milliliters of sterile distilled water and incubate for 30 minutes. After confirming the cells are lysed, collect the suspension into a 1.5 milliliter tube. Centrifuge the suspension at 6, 000 times G for five minutes at room temperature and discard the supernatant carefully before resuspending the pellet in 10 microliters of sterile distilled water.
Inoculate 10 microliters of the suspension onto the PDA plate and incubate at 28 degrees Celsius in the dark. At the beginning of the phagocytosis process, free conidia, conidia attached to the outer membrane of macrophages or completely internalized by macrophages were observed. With increased phagocytosis time, free and conidia attached to the outer membrane were completely internalized by the macrophages.
The conidia were also observed in an upper plane compared to the macrophages, and more than one conidium was found in the same macrophage. After long periods, phagocytosed conidia germinate inside the macrophages and free conidia germinate in the R10 medium forming hyphae at 37 degrees Celsius. The phagocytosed conidia remained viable even after 120 hours of interaction with the macrophages.
After phagocytosis, growth was monitored in terms of the time required for the culture to occupy the entire plate and to form colored green conidia.
