This work was supported by the following Brazilian financing institutions: Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado da Bahia (FAPESB) with grants RED0011/2012 and RED008/2014. U.R.S., J.O.C., and M.E.S.M. acknowledge the scholarship granted by Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior (CAPES) and FAPESB, respectively.

Ethical considerations and human subjects 
All experiments with humans described in this study were conducted according to the Declaration of Helsinki and Brazilian Federal laws and approved by the State University of Santa Cruz&#39;s Ethics Committee (project identification code: 550.382/ 2014).
Human peripheral blood was collected from healthy volunteers from Ilh&#233;us city, Bahia, Brazil, not exposed to occupational activities related to the studied fungus. Individuals with reported health medical conditions or using medication were excluded. All subjects voluntarily agreed to participate and signed an informed consent form before their inclusion in this study.
1. Preparation of the reagents and solutions
NOTE: Prepare the following reagents and solutions before proceeding. See the Table of Materials (TOM) for the reagent and material supplier information.
<ol>
	<li>Prepare a balanced sterile phosphate-buffered saline solution 1x (PBS). 
	NOTE: In this protocol, endotoxin-free PBS was not used.</li>
	<li>Prepare potato dextrose agar (PDA) medium, and pour 20 mL into each Petri dish for the T. stromaticum culture. Prepare six plates with PDA for each donor, and use one plate for each condition: 3 h, 24 h, 48 h, 72 h, 96 h, and 120 h. Additionally, use three plates for each control: three for the Trichoderma control and three for the R10 medium control. Use Petri dishes of 60 mm x 15 mm or 100 mm x 15 mm for optimal conidia recovery.</li>
	<li>Sterilize 100% glycerol.</li>
	<li>Prepare an R10 medium for the cell culture: RPMI medium supplemented with 10% fetal bovine serum and 1% antibiotic (penicillin/streptomycin).</li>
	<li>Sterilize distilled water for the cell lysis.</li>
	<li>Prepare water at pH 7.0 for the coloration process. 
	NOTE: If necessary, adjust the pH using a pH meter and solutions of 0.1 M/L NaOH and 0.1 M/L HCl.&#160;</li>
	<li>Sterilize round glass coverslip (13 mm) and tweezers.</li>
</ol>
2. Trichoderma stromaticum culture and processing
<ol>
	<li>Preparation of a mother stock (M1) of T. stromaticum: 
	NOTE: A mother stock (M1) is required to ensure that all the experiments are done from the culture of the same fresh generation (F1).
	<ol>
		<li>Grow a T. stromaticum inoculum&#160;in Petri dishes with PDA at 28 &#176;C in the dark for 7-10 days until the conidia are observed and the culture turns green.</li>
		<li>Wash the culture of T. stromaticum.&#160;Add 3-5 mL of PBS to the culture surface using a pipette. Collect the PBS from the plate, and again dispense it over the culture to gradually remove the conidia using the pipette. Repeat this process as much as necessary until the suspension turns dark green. 
		NOTE: Alternatively, gentle washing of the culture by adding 3-5 mL of PBS to the plate followed by performing a circular movement is suggested until the suspension turns green to recover the conidia. With repeated pipetting, more conidia can be collected.</li>
		<li>Transfer the recovered suspension from the plate into a 15 mL sterile tube with a pipette. Repeat steps 2.1.2-2.1.3 as many times as necessary to obtain more conidia.</li>
		<li>Centrifuge the suspension at 1,160 x g for 5 min at 12 &#176;C, and resuspend the pellet in 5 mL of PBS. Repeat this step (2.1.4) three times to obtain a hyphae-free suspension. 
		NOTE: To resuspend the pellet properly, after centrifugation, brief vortexing is recommended.</li>
		<li>Resuspend the final pellet in 2-3 mL of PBS, and count the conidia in a Neubauer chamber using a dilution of 1:100 or 1:1,000. 
		NOTE: In optimal conditions, a final concentration of 2 x 108 conidia/mL can be recovered from the culture. Assessing the conidia viability using the trypan blue exclusion method is optional.</li>
		<li>Aliquot the suspension into 1.5 mL sterile tubes by adding 0.5 mL of the conidia suspension (from step 2.1.5) to each tube. Then, add 0.5 mL of sterile 100% glycerol to obtain M1 stocks. Vortex briefly, identify the tubes, and store at &#8722;20 &#176;C or &#8722;80 &#176;C.</li>
	</ol>
	</li>
	<li>Culture of T. stromaticum and obtaining conidia for the experiment
	<ol>
		<li>Plate 10 &#181;L of the M1 suspension (prepared in step 2.1) in PDA at 28 &#176;C in the dark for 7-10 days until the conidia are observed and the culture turns green to obtain the fresh F1 generation. 
		NOTE: To ensure a fresh T. stromaticum culture for the experiment, assess the time required for fungus growth and the time required for macrophage differentiation.</li>
		<li>Collect the conidia, repeating steps 2.1.2-2.1.4.</li>
		<li>Resuspend the final pellet in 2-3 mL of PBS. Assess the conidia viability using the trypan blue exclusion method, or count the conidia as mentioned in step 2.1.5.</li>
	</ol>
	</li>
</ol>
3. Peripheral blood mononuclear cell (PBMC) isolation
NOTE: The PBMCs are obtained by the density barrier method using a polysucrose solution of density 1.077 g/mL30.
<ol>
	<li>Aliquot 15 mL of the polysucrose solution to one 50 mL tube for every donor sample that will be collected.</li>
	<li>Collect 20 mL of blood from each donor using three to four EDTA tubes. Recruit a minimum of four donors per experiment. Do not pool the blood of the donors; instead, use each sample separately as a biological replicate. For each donor, transfer the blood to one 50 mL tube, and add PBS to obtain a final volume of 35 mL.</li>
	<li>Transfer the blood suspension (step 3.2) slowly along the wall of the tube containing the polysucrose solution (step 3.1) to form a biphasic suspension. 
	NOTE: To prevent the mixing of the blood with the polysucrose solution, add the blood slowly. The use of a minimum of 7 mL of blood from each donor for the PBMC isolation is recommended.</li>
	<li>Immediately centrifuge the tube containing the biphasic suspension at 1,600 x g for 30 min at 24 &#176;C. Program the centrifugation of the PBMCs: use acceleration 1 and deceleration 0 to avoid an abrupt stop of the centrifugation.</li>
	<li>Observe the formation of a white ring containing the PBMCs after centrifugation. The red blood cells and granulocytes reside in the lower layer, the next layer contains the polysucrose solution, and the PBMCs reside in the white ring between the polysucrose solution and the plasma (upper layer). Collect the white ring by aspirating gently using a pipette or a sterile Pasteur pipette so as not to catch red blood cells, plasma, or polysucrose solution, and transfer to one 15 mL tube.</li>
	<li>Add PBS to the 15 mL tube containing the PBMCs to obtain a final volume of 10 mL. Homogenize the suspension, and centrifuge the tube at 1,600 x g for 30 min at 24 &#176;C.</li>
	<li>Wash the cells three times at 1,600 x g for 5 min at 24 &#176;C with 10 mL of PBS. Then, resuspend the final pellet in 2 mL of R10 medium.</li>
	<li>Assess the cell viability using the trypan blue exclusion method, and count the cells in a Neubauer chamber.</li>
</ol>
4. Phagocytosis kinetics
NOTE: The human peripheral blood mononuclear-derived macrophages are treated with T. stromaticum conidia at a multiplicity of infection (MOI) of 1:10 or only R10 medium as a control. The multiplicity of infection (MOI) represents the ratio of the infectious agents added to the infection targets and is presented as absolute numbers: MOI = agents:targets31. Here, we use 1 T. stromaticum conidia (agent) for 10 macrophages (targets). Then, the cells are incubated at 37 &#176;C and 5% CO2 for different periods of time (3 h, 24 h, 48 h, 72 h, 96 h, and 120 h). Next, a round glass coverslip is used to visualize the adherent macrophages involved in the phagocytosis process under a microscope. An experimental design figure is provided (Figure 1).
NOTE: The use of 24-well plates is suggested.
<ol>
	<li>Treatment of human-derived macrophages with T. stromaticum conidia
	<ol>
		<li>Add a sterile round glass coverslip (13 mm) to each well using sterile tweezers.</li>
		<li>Adjust the volume of the cell suspension obtained in steps 3.7-3.8 to plate 8 x 105 cells per well to a final volume of 1 mL with R10 medium. Use an inverted microscope to assess the cell morphology. 
		NOTE: The cells must be suspended in the medium and have a rounded morphology.</li>
		<li>Incubate the cells at 37 &#176;C under a 5% CO2 atmosphere for 7 days, and replace the medium with fresh R10 medium every 2 days for the differentiation of the monocytes into macrophages32. Observe the cell morphology using an inverted microscope. The macrophages will present spindled morphology, with flatted and extended shapes; an increased cytoplasmatic ratio and the presence of pseudopodia can also be observed. Look for adherent mononuclear-derived macrophages. 
		NOTE: A very small proportion of undifferentiated monocytes may remain suspended in the medium.</li>
		<li>Remove the medium of each well using a pipette, and wash the cells with 1 mL of PBS to remove debris and non-adherent cells.</li>
		<li>Use the suspension prepared in step 2.2 to prepare a new suspension of T. stromaticum conidia at a final concentration of 8 x 104 conidia/mL in R10 medium.</li>
		<li>Add 1 mL of the conidia suspension prepared in step 4.1.5 to each treatment well (3 h, 24 h, 48 h, 72 h, 96 h, and 120 h) to obtain a&#160;MOI of 1:10. To the controls, add only 1 mL of R10 medium to the well.</li>
		<li>Challenge the macrophages for 3 h with T. stromaticum conidia at 37 &#176;C under a 5% CO2 atmosphere. After 3 h, remove the medium, and wash the cells to remove the non-phagocytosed conidia.</li>
		<li>Add 1 mL of R10 medium to each well, and incubate the plate for different times (3 h, 24 h, 48 h, 72 h, 96 h, and 120 h) at 37 &#176;C under a 5% CO2 atmosphere.</li>
	</ol>
	</li>
	<li>Coloration and analysis
	<ol>
		<li>Remove the medium from each well after the corresponding incubation (step 4.1.8) is complete, and add 1 mL of PBS to that well.</li>
		<li>Remove the round glass coverslips from the wells using sterile tweezers and a needle for coloring with a staining kit. 
		NOTE: Alternatively, staining can be done using May Gr&#252;nwald-Giemsa stain33.</li>
		<li>To each of the round glass coverslips, add stain fixator (5 s), Dye 1 (5 s), Dye 2 (2 s), and buffered water pH 7.0 (5 s), which are the components of the staining kit. Wait until they are dry, and fix the coverslips on a glass slide using mounting agent.</li>
		<li>Quantify the result: Count a total of 100 macrophages (M&#248;) for each time condition. Using an optical microscope, count the number of macrophages with phagocytosed conidia using a 100x objective and immersion oil (equation [1]). 
		<img alt="Equation 1" src="/files/ftp_upload/65231/65231eq01v2.jpg" style="margin: auto;" /> (1)</li>
		<li>Obtain the number of conidia phagocytosed per macrophage: Correlate the conidia present in 100 macrophages divided by the number of macrophages with at least one phagocytosed conidium (equation [2])34. 
		<img alt="Equation 2" src="/files/ftp_upload/65231/65231eq02v2.jpg" style="margin: auto;" /> (2) 
		&#8203;NOTE: To evaluate and count the conidia, the ImageJ software can be used35.</li>
		<li>Photograph the phagocytosis from each sample and time condition using 40x and 100x objectives to present the results.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65231/65231fig01.jpg" /> 
Figure 1: Schematic representation of the phagocytosis and conidial viability assay using human peripheral blood mononuclear-derived macrophages. (1-10) Phagocytosis assay: The T. stromaticum must be grown in PDA, and the conidia are recovered by washing the plate with PBS.&#160;The&#160;PBMCs should be isolated by the density barrier method using the described protocol, cultured in 24-well plates with sterile round glass coverslips for 7 days for differentiation into macrophages, and then treated with an MOI of 1:10 with different time intervals. The round glass coverslips are removed and stained with the staining kit, and the results are analyzed using light microscopy. (1-8,11-13) Conidia viability assay: After isolation, the PBMCs are cultured in 24-well plates without round glass coverslips for 7 days for differentiation into macrophages and then treated with an MOI of 1:10 for different time intervals. The cells must be lysed by incubation with distilled water; the suspension is then centrifuged, and then the resuspended pellet is plated in PDA to analyze the growth kinetics of Trichoderma. This figure was designed using a database of images. <a href="https://www.jove.com/files/ftp_upload/65231/65231fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Conidial viability assay after phagocytosis
NOTE: An experimental design figure is provided (Figure 1).
NOTE: Use 24-well plates.
<ol>
	<li>Challenge the human-derived macrophages with T. stromaticum conidia.
	<ol>
		<li>Repeat steps 4.1.2-4.1.8 to differentiate the monocytes, and treat the macrophages with T. stromaticum conidia. 
		NOTE: Do not use round glass coverslips in the wells. Use the same time intervals used for assaying the phagocytosis kinetics so that the phagocytosis can be correlated with the conidial viability.</li>
		<li>Evaluate the impact of the R10 medium on the T. stromaticum growth by using wells with the suspension prepared in step 4.1.5 (R10 medium + conidia) as a control.</li>
	</ol>
	</li>
	<li>Conidial viability assay
	<ol>
		<li>Remove the medium after each incubation period (3 h, 24 h, 48 h, 72 h, 96 h, and 120 h), and wash the cells twice with PBS to remove the conidia that have not been phagocytosed. 
		NOTE: The control containing R10 medium + conidia should not be washed.</li>
		<li>Add 0.5 mL of sterile distilled water to each well, and incubate the plate at 37 &#176;C and 5% CO2 for 30 min. After this time, observe the cell morphology under an inverted microscope to ensure the cells have been lysed.</li>
		<li>Collect the suspension, and transfer it to 1.5 mL tubes. Centrifuge the suspension using a mini centrifuge at 6,000 x g for 5 min at room temperature.</li>
		<li>Discard the supernatant carefully, resuspend the pellet in 10 &#181;L of sterile distilled water, and inoculate 10 &#181;L of the suspension into the PDA-containing plate at 28 &#176;C in the dark.</li>
		<li>Prepare a positive control for T. stromaticum growth: Use the suspension of T. stromaticum conidia (step 2.2) to prepare a new suspension with a final concentration of 8 x 106 conidia/mL. Plate 10 &#181;L of this suspension in PDA at 28 &#176;C in the dark. 
		NOTE: 10 &#181;L of this suspension will contain the same number of conidia (8 x 104) that was used to infect the blood mononuclear-derived macrophages. The difference between this positive control and the one mentioned in step 5.1.2 is that the control mentioned above in step 5.1.2 is used to evaluate the possible impact of the R10 medium on Trichoderma growth during the time of phagocytosis. This positive control in step 5.2.5 using the suspension of T. stromaticum conidia collected in PBS represents a control for Trichoderma growth without the influence of R10 medium to reflect how the fungus grows naturally. Using this control, it is possible to evaluate if the R10 medium influences the Trichoderma growth.</li>
		<li>Observe the growth kinetics of T. stromaticum in PDA every day, and photograph the culture.</li>
		<li>Analysis: Two points can be used to evaluate the results: 1) the time (in days) required for the culture to occupy the entire culture plate, and 2) the time (in days) until the appearance of the characteristic green pigmentation of the T. stromaticum conidia in culture.</li>
	</ol>
	</li>
</ol>
6. Statistical analysis
<ol>
	<li>Use a Kruskal-Wallis test to determine statistically significant differences between the 8 groups/treatments. Consider p &#60; 0.05 as statistically significant. All the data are presented as means &#177; standard deviation (SD). In the figures, # indicates statistical significance at p &#60; 0.05.</li>
</ol>

Fungal Clearance Efficiency of Macrophages

All the authors declare no conflicts of interest.

For several fungal pathogens including Aspergillus fumigatus, Cryptococcus, Candida albicans,&#160;and others, conidial or yeast phagocytosis is a key process that allows for the investigation of the cytoplasmic and molecular events in macrophage-pathogen interactions, as well as for the determination of the time of death of the internalized conidia14,39,40. Phagocytosis is the key process in the T...

The Trichoderma genus (Order: Hypocreales, Family: Hypocreaceae) is composed of ubiquitous, saprophytic fungi that are parasites of other fungal species and are capable of producing a range of commercially useful enzymes1. These fungal species are used for the production of heterologous proteins2, the production of cellulose3, ethanol, beer, wine, and paper4, in the textile industry5, food industry6, and in agriculture as biological control agents7,8. In addition to the industrial interest in these fungal species, the increasing number of infections in humans has given some Trichoderma species the status of opportunistic pathogens9.
Trichoderma spp. grow rapidly in culture, with initially white and cottony colonies that turn greenish yellow to dark green10. They are adapted to live in a wide range of pH and temperature conditions, and the opportunistic species are able to survive at physiological pH and temperatures and, thus, colonize different human tissues11,12,13. Importantly, the rise in the infection rate of Trichoderma spp. may be associated with virulence factors, and these are not well studied. In addition, studies focusing on understanding the immune response against opportunistic Trichoderma species are still rare.
During an infection, along with neutrophils, macrophages represent the line of defense responsible for phagocytosis, and, thus, prevent the growth and colonization of pathogens in different tissues. Using pattern recognition receptors, such as Toll-like receptors and C-type lectin receptors, macrophages phagocytose fungi and process them into phagolysosomes, thus promoting a respiratory burst, the release of pro-inflammatory cytokines, and the destruction of the phagocytosed microorganisms14. The mechanism of phagocytosis, however, can be affected and evaded by different microbial strategies, such as the size and shape of the fungal cells; the presence of capsules that hinder phagocytosis; decreasing the number of phagocytosis-inducing receptors; the remodeling of the structure of actin fibers in the cytoplasm; hindering the formation of pseudopodia; and phagosome or phagolysosome escape after the phagocytosis process14.
Many pathogens, including Cryptococcus neoformans, use macrophages as a niche to survive in the host, disseminate, and induce infection15. The phagocytosis and clearance assay is used to evaluate the immune response against pathogens and to identify the microbial strategies employed to evade the innate immune system15,16,17. This type of technique can also be used to examine the differential kinetics of phagocytosis, delayed phagosome acidification, and oxidative burst that result in reduced fungal killing18.
Different methods can be used to evaluate phagocytosis, fungal survival, and the evasion of the phagosome maturation process. These include fluorescence microscopy, which is used to observe phagocytosis, the cellular location, and the molecules produced during phagocytosis19; flow cytometry, which provides quantitative data on phagocytosis and is used to evaluate the different markers involved in the process20,21; intravital microscopy, which is used to assess microbial capture and phagosome maturation22; antibody-mediated phagocytosis, which is used to assess the specificity of the phagocytosis process for a pathogen23; and others24,25,26,27.
The protocol presented here employs a common, low-cost, and direct method using an optical microscope and plate growth assay to assess the phagocytosis and killing of fungal conidia. This protocol will provide the readers with step-by-step instructions for performing the phagocytosis and clearance assay using human peripheral blood mononuclear-derived macrophages exposed to T. stromaticum. PBMCs were used because Trichoderma conidia are applied as a biocontrol against phytopathogens and a biofertilizer for plant crops worldwide and have caused several human infections, called Trichodermosis. Besides that, there are only two previous works focusing on the interaction between Trichoderma conidia and the human immune system, in which we examined neutrophils28 and autophagy in macrophages29. This article shows first how the phagocytosis of the conidia of T. stromaticum by PBMC-derived macrophages can be studied, and then how the viability of the engulfed conidia can be assessed using simple microscopy-based techniques. This protocol may further facilitate investigations on macrophage-associated immune response or immune system modulation-related mechanisms.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Corning</td>
			<td>CLS431470</td>
			<td>15 mL centrifuge tubes, polypropylene, conical bottom with lid, individually sterile</td>
		</tr>
		<tr>
			<td>24-Well Flat Bottom Cell Culture Plate</td>
			<td>Kasvi</td>
			<td>K12-024</td>
			<td>Made of polystyrene with alphanumeric identification; The Cell Culture Plate is DNase, RNase and pyrogen-free and free of cytotoxic substances; Sterilized by gamma radiation;</td>
		</tr>
		<tr>
			<td>Cell culture CO2 incubator</td>
			<td>Sanyo</td>
			<td>303082</td>
			<td>A CO2 incubator serves to create and control conditions similar to a human body, thus allowing the in vitro growth and proliferation of different cell types.</td>
		</tr>
		<tr>
			<td>Centrifuge Microtube (eppendorf type) 1.5 mL</td>
			<td>Capp</td>
			<td>5101500</td>
			<td>Made from polypropylene, with a cap attached to the tube for opening and closing with just one hand. It has a polished interior against protein adhesion and for sample visibility, being free of DNase, RNase and Pyrogens</td>
		</tr>
		<tr>
			<td>Circular coverslip 15 mm</td>
			<td>Olen</td>
			<td>K5-0015</td>
			<td>Circular coverslips are used for microscopy techniques in cell culture. Made of super transparent translucent glass; with thickness of 0.13 mm</td>
		</tr>
		<tr>
			<td>Class II Type B2 (Total Exhaust) Biosafety Cabinets</td>
			<td>Esco Lifesciences group</td>
			<td>2010274</td>
			<td>Airstream Class II Type B2 Biosafety Cabinets (AB2) provide product, operator and environmental protection and are suitable for work with trace amounts of toxic chemicals and agents assigned to biological safety levels I, II or III. In a Class II Type B2 cabinet, all inflow and downflow air is exhausted after HEPA/ULPA filtration to the external environment without recirculation across the work surface.</td>
		</tr>
		<tr>
			<td>Dextrose Potato Agar medium</td>
			<td>Merck</td>
			<td>145</td>
			<td>Potato Dextrose Agar is used in the cultivation and enumeration of yeasts and fungi</td>
		</tr>
		<tr>
			<td>EDTA vacuum blood collection tube</td>
			<td>FirstLab</td>
			<td>FL5-1109L</td>
			<td>EDTA is the recommended anticoagulant for hematology routines as it is the best anticoagulant for preserving cell morphology.</td>
		</tr>
		<tr>
			<td>Entellan</td>
			<td>Merck</td>
			<td>1.07961&#160;</td>
			<td>Fixative agent; Entellan is a waterless mounting medium for permanent mounting for microscopy.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>A2720801</td>
			<td>Fetal bovine serum (FBS) is a&#160;universal growth supplement of cell and tissue culture media. FBS is a natural cocktail of most of the factors required for cell attachment, growth, and proliferation, effective for most types of human and animal (including insect) cells.</td>
		</tr>
		<tr>
			<td>Flaticon</td>
			<td></td>
			<td></td>
			<td>&#160;database of images</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Merck</td>
			<td>24900988</td>
			<td>The cryoprotectant agent glycerol is used for freezing cells and spores</td>
		</tr>
		<tr>
			<td>Histopaque-1077</td>
			<td></td>
			<td></td>
			<td>polysucrose solution</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td></td>
			<td></td>
			<td>&#160;Image analysis software</td>
		</tr>
		<tr>
			<td>Microscopy slides</td>
			<td>Precision</td>
			<td>7105</td>
			<td>Slide for Microscopy 26 x 76 mm Matte Lapped Thickness 1.0 to 1.2 mm. Made of special optical glass and packaged with silk paper divider with high quality transparency free of imperfections</td>
		</tr>
		<tr>
			<td>Mini centrifuge</td>
			<td>Prism</td>
			<td>C1801</td>
			<td>The Prism Mini Centrifuge was designed to be extremely compact with an exceptionally small footprint. Includes 2 interchangeable quick-release rotors that spin up to 6000 rpm. An electronic brake provides quick deceleration and the self-opening lid allows easy access to the sample, reducing handling time.</td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Kasvi</td>
			<td>K5-0011</td>
			<td>The Neubauer Counting Chamber is used for counting cells or other suspended particles.</td>
		</tr>
		<tr>
			<td>Panoptic fast&#160;</td>
			<td>Laborclin</td>
			<td>620529</td>
			<td>Laborclin&#39;s&#160; panoptic fast c is a kit for quick staining in hematology</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin Solution - 10,000U</td>
			<td>LGC- Biotechnology&#160;</td>
			<td>BR3011001</td>
			<td>antibiotic is used in order to avoid possible contamination by manipulation external to the laminar flow.</td>
		</tr>
		<tr>
			<td>Petri dish 90 x 15 mm Smooth</td>
			<td>Cralplast</td>
			<td>18248</td>
			<td>Disposable Petri dish; Made of highly transparent polystyrene (PS); flat bottom; Smooth;Size: 90 x 15 mm.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>thermo fisher Scientific</td>
			<td>10010001</td>
			<td>PBS is a water-based saline solution with a simple formulation. It is isotonic and non-toxic to most cells. It includes sodium chloride and phosphate buffer and is formulated to prevent osmotic shock while maintaining the water balance of living cells.</td>
		</tr>
		<tr>
			<td>Pipette Pasteur 3 mL Sterile</td>
			<td>Accumax</td>
			<td>AP-3-B-S</td>
			<td>STERILE ACCUMAX PASTEUR 3 ML PIPETTE with 3 mL capacity, made of transparent low-density polyethylene (LDPE) and individually sterile</td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>TS-HM16R</td>
			<td>The Thermo Scientific Heraeus Megafuge 16R Refrigerated Centrifuge is a refrigerated centrifuge with the user-friendly control panel makes it easy to pre-set the speed, RCF value, running time,&#160;temperature, and running profile. The Megafuge 16R can reach maximum speeds of&#160;15,200 RPM and maximum RCF of 25,830 x g.</td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium</td>
			<td>Merck</td>
			<td>MFCD00217820</td>
			<td>HEPES Modification, with L-glutamine and 25 mM HEPES, without sodium bicarbonate, powder, suitable for cell culture</td>
		</tr>
		<tr>
			<td>The single channel micropipettes</td>
			<td>Eppendorf</td>
			<td>Z683809</td>
			<td>Single-channel micropipettes are used to accurately transfer and measure very small amounts of liquids.</td>
		</tr>
		<tr>
			<td>Tip for Micropipettor</td>
			<td>Corning</td>
			<td>4894</td>
			<td>Capacity of 10 &#181;L and 1,000 &#181;L Autoclavable</td>
		</tr>
		<tr>
			<td>Triocular inverted microscope</td>
			<td>LABOMED</td>
			<td>VU-7125500</td>
			<td>It allows you to observe cells inside tubes and bottles, without having to open them, thus avoiding contamination problems.</td>
		</tr>
	</tbody>
</table>

viability assay of trichoderma stromaticum conidia inside human peripheral blood mononuclear-derived macrophages

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial phagocytosis is a key process that allows for the investigation of the cytoplasmic and molecular events involved in macrophage-pathogen interactions, as well as for the determination of the time of death of internalized conidia. The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The evasion of phagocytosis and escape of phagosomes are mechanisms of fungal virulence. Here, we report the methods that can be used for the analysis of the phagocytosis, clearance, and viability of T. stromaticum conidia, a fungus which is used as a biocontrol and biofertilizer agent and is capable of inducing human infections. The protocol consists of 1) Trichoderma culture, 2) washing to obtain conidia, 3) the isolation of peripheral blood mononuclear cells (PBMCs) using the polysucrose solution method and the differentiation of the PBMCs into macrophages, 4) an in vitro phagocytosis method using round glass coverslips and coloration, and 5) a clearance assay to assess the conidia viability after conidia phagocytosis. In summary, these techniques can be used to measure the&#160;fungal clearance efficiency of macrophages.

Author Spotlight: Unraveling the Interplay Between &lt;em&gt;Trichoderma stromaticum&lt;/em&gt; and the Mammalian Immune System

Viability Assay of Trichoderma stromaticum Conidia Inside Human Peripheral Blood Mononuclear-Derived Macrophages

Our research is focused on understand the interaction between the Trichoderma fungi and the mammalian immune system. How do some Trichoderma species cause disease, how can they modulate the immune system, and how can this modulation impact the development of the disease? To answer this question, we use in vitro randomized models.We show that Trichoderma species modulates macrophages and neutrophils by targeting receptors involved in the phagocytosis process. These receptors include toll-like receptors and lectin receptors. When there is a decrease in phagocyte capacity of immune cells, susceptibility to pathogens, especially intracellular pathogens does increase.Ongoing, have many invasion mechanism to survive in the host. This include avoiding phagocytosis and survive inside macrophage. Pathogenic species use macrophage as Trojan horses to survive in disseminating the host.Here, we propose that to know if Trichoderma can survive inside macrophage, and for how long they survive.

The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. We used the phagocytosis of T. stromaticum conidia to assess the viability of the conidia after phagocytosis, since the evasion of phagocytosis and the escape of phagosomes are mechanisms of fungal virulence. Researchers should perform these techniques as one of the first assays when investigating a species of clinical interest.
<p class="jove_c...

Watch this Scientific Journal Video about Author Spotlight: Unraveling the Interplay Between Trichoderma stromaticum and the Mammalian Immune System at JoVE.com

The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The purpose of this manuscript is to present a method for evaluating the phagocytosis and clearance abilities of human peripheral blood mononuclear-derived macrophages stimulated with Trichoderma stromaticum conidia.

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial ...

viability-assay-trichoderma-stromaticum-conidia-inside-human

Research

JoVE Journal

Immunology and Infection

471 Views.  State University of Santa Cruz - UESC. Our research is focused on understand the interaction between the Trichoderma fungi and the mammalian immune system. How do some Trichoderma species cause disease, how can they modulate the immune system, and how can this modulation impact the development of the disease? To answer this question, we use in vitro randomized models.We show that Trichoderma species modulates macrophages and neutrophils by targeting receptors involved in the phagocytosis process. These receptors include tol...

Video: Author Spotlight: Unraveling the Interplay Between Trichoderma stromaticum and the Mammalian Immune System

Culturing Trichoderma stromaticum and Obtaining Conidia for Phagocytosis Assay

To begin, plate 10 microliters of the mother stock suspension of Trichoderma stromaticum on the PDA plate. Incubate the culture at 28 degrees Celsius in the dark for seven to 10 days until the conidia are observed and the culture turns green to obtain the fresh F1 generation. Wash the culture by adding three to five milliliters of PBS to the plate, then collect and again dispense the PBS over the culture to gradually remove the conidia.Repeat this process until the suspension turns dark green. Transfer the recovered suspension from the plate into a sterile 15-milliliter tube. Centrifuge the suspension at 1, 160 G for five minutes at 12 degrees Celsius and resuspend the pellet in five milliliters of PBS.Resuspend the final pellet in two to three milliliters of PBS and count the conidia in a Neubauer chamber.

Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from Human Blood and Differentiation of PBMCs into Macrophages for Phagocytosis Assay

Transfer the blood collected from the donor to a 50 milliliter tube and add PBS to obtain a final volume of 35 milliliters. Transfer the blood slowly along the wall of the tube containing the polysucrose solution to form a biphasic suspension. Immediately, centrifuge the suspension at 1, 600 G for 30 minutes at 24 degrees Celsius.After centrifugation, observe the formation of a white ring containing the PBMC's, and collect it by aspirating gently in a 15 milliliter tube. Then, add PBS to the tube containing the PBMCs to obtain a final volume of 10 milliliters. Homogenize the suspension in centrifuge at 1, 600 G for 30 minutes at 24 degrees Celsius.After centrifugation, wash the cells three times at 1, 600 G for five minutes with 10 milliliters of PBS. Before resuspending the pellet in two milliliters of R10 medium.

Phagocytosis Kinetics and Trichoderma stromaticum Conidial Viability Assay Using Human Peripheral Blood Mononuclear-Derived Macrophages

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.