The authors wish to acknowledge Jos&#233; Luis Ramos-Balderas for his technical support. This work was supported by the following grant to PLL from CONACYT (FORDECYT-PRONACE-303027). OM-P and EO-M received a fellowship from CONACYT (736162 and 481437, respectively). MCM-M received a fellowship from CONACYT (Estancias Posdoctorales por M&#233;xico 2022 (3)).

All animal experiments described here were approved by the Internal Committee for Animal Handling (CICUAL) of the Institute of Cellular Physiology, National Autonomous University of Mexico.
NOTE: A flowchart of the entire protocol is shown in Figure 1.
1. Housing of mice
<ol>
	<li>Use 8-10-week-old, female or male groups of mice, housed in animal facilities with constant temperature and humidity in 12 h light/dark cycles, with ad libitum access to water and food. 
	NOTE: This protocol uses an IL-9 reporter mouse strain in C57BL/6 background, as previously described4; however, other IL-9 reporter strains could be used6,7,8, as well as intracellular IL-9 staining, with variable results9.</li>
</ol>
2. Infection of mice
<ol>
	<li>Shave the back of the mice from the middle of the body to near the base of the tail 1 day prior to infection. The use of anesthetics is not essential.</li>
	<li>Subcutaneously inoculate each mouse with 200 viable third-stage N. brasiliensis larvae (L3) in 100 &#181;L of phosphate buffered saline (PBS)10 in the lower back, as previously described2,11, or inject 100 &#181;L of PBS alone as a control. Sacrifice the animals on day 4, day 7, or day 10 post-infection.</li>
</ol>
3. Isolation of lung, small intestine, mediastinal, and mesenteric lymph nodes
<ol>
	<li>Euthanize the mouse by cervical dislocation. Place the mouse on its back and spray it with 70% ethanol. Make a midline incision using scissors and open the skin to expose the abdominal and thoracic areas. 
	NOTE: Isoflurane can be used as an alternative euthanasia method12. Carbon dioxide chambers are not recommended, as CO2 leads to lung tissue damage and hemorrhage13.</li>
	<li>Isolation of mediastinal lymph nodes and lungs
	<ol>
		<li>Make an incision at the sternum and cut in a &#34;V&#34; shape to remove the ribs and thoracic muscles. Once the thoracic cavity is exposed, locate the mediastinal lymph nodes next to the esophagus, below the heart (see Supplementary Figure 1).</li>
		<li>Extract the mediastinal lymph nodes and collect them in 1 mL of R-10 medium (Table 1) in a 12-well culture plate. Keep on ice, protected from light until processing. 
		NOTE: The samples should be protected from light, to avoid decreasing the fluorescent signal from the reporter mice used in these experiments.</li>
		<li>Extract the lungs and collect them in 1 mL of R-10 medium in a 12-well culture plate per mouse. Keep on ice, protected from light until processing.</li>
	</ol>
	</li>
	<li>Isolation of mesenteric lymph node chains and small intestines
	<ol>
		<li>Expose the peritoneal cavity, and carefully move the small intestine to the right to expose the mesenteric lymph node (MLN) chain along the colon.</li>
		<li>Using forceps, remove the MLN, roll it gently on a paper towel, and pull the fat off.</li>
		<li>Transfer the MLN to 1 mL of R-10 medium in a 12-well culture plate. Keep on ice, protected from light until processing.</li>
		<li>Cut the small intestine just below the pyloric sphincter and above the cecum. Pull the intestine out slowly with the help of forceps, removing the attached mesentery and fat tissue.</li>
		<li>Place the small intestine on a paper towel and generously moisten it with PBS. Remove the Peyer&#39;s patches from the small intestine with scissors. 
		NOTE: To maintain viability, remove the remaining fat tissue, and keep the small intestine moist with PBS during the entire process.</li>
		<li>Cut the small intestine longitudinally using scissors, and gently slide the forceps over the open intestine to remove the fecal content and mucus.</li>
		<li>Hold the small intestine with forceps, and wash it in 5 mL of PBS on ice by carefully submerging it a few times. Repeat twice.</li>
		<li>Cut the small intestine into short pieces (approximately 5 mm), and collect them in a 50 mL conical tube with 10 mL of HBSS14 with 2% FBS (Table 1). Immediately continue to isolate intraepithelial and lamina propria cells.</li>
	</ol>
	</li>
</ol>
4. Preparation of single-cell suspensions from the small intestine, lung, and lymph nodes
NOTE: It is extremely important to process a maximum of six mice per person when preparing single-cell suspensions from small intestines, as cell viability decreases significantly with longer processing periods. This method was adapted from the Heligmosomoides polygyrus infection mouse model5.
<ol>
	<li>Pre-warm the shaking incubator, R-20 medium, HBSS, and HBSS-2 mM EDTA (Table 1) at 37 &#176;C.</li>
	<li>Prepare 10 mL of small intestine digestion medium (Table 1) per sample.</li>
	<li>Shake the intestine pieces from step 3.3.8 vigorously by hand.</li>
	<li>Filter each sample through a nylon mesh (approximately 10 cm x 10 cm) over a glass funnel. Wash the sample by adding 10 mL of pre-warmed HBSS over the mesh and discard the flow-through. Repeat one more time. 
	NOTE: Use a new mesh for each sample and reuse it throughout the procedure.</li>
	<li>Remove the mesh from the funnel, and collect the sample from the mesh in a 50 mL conical tube with 10 mL of warm HBSS-2 mM EDTA (step 4.1). Incubate for 10 min at 37 &#176;C, with shaking at 200 rpm.</li>
	<li>Vortex for 10 s at maximum speed (3,200 rpm) and filter the sample with the mesh over a glass funnel, recovering the flow-through in a 50 mL conical tube.</li>
	<li>Repeat steps 4.5 and 4.6 twice, recovering the flow-through in the same 50 mL conical tube. The intraepithelial cells are located in this 30 mL fraction. Save the remaining tissue.</li>
	<li>Single-cell suspension preparation from intraepithelial cells
	<ol>
		<li>Centrifuge the 30 mL of cell suspension, recovered in step 4.7, at 450 x g for 5 min at room temperature (RT). Discard the supernatant.</li>
		<li>Add 5 mL of PBS and centrifuge at 450 x g for 5 min at RT. Discard the supernatant.</li>
		<li>Resuspend the cell pellet in 3 mL of RPMI 10% FBS-20 &#181;g/mL DNase (Table 1). Keep on ice, protected from light until cell staining.</li>
	</ol>
	</li>
	<li>Single-cell suspension preparation from lamina propria cells
	<ol>
		<li>Wash the remaining small intestine tissue from step 4.7 by pouring over 10 mL of warm HBSS through the mesh over the funnel. Repeat the wash. Collect the tissue from the mesh in a 50 mL conical tube with 10 mL of small intestine digestion medium.</li>
		<li>Incubate for 30 min at 37 &#176;C, with shaking at 200 rpm. Vortex at maximum speed for 10 s every 5 min.</li>
		<li>Add 10 mL of FACS-EDTA buffer (Table 1) to stop the digestion reaction, and place it on ice.</li>
		<li>Filter each sample through a 100 &#181;m cell strainer using a serological pipette, recovering the suspension in a 50 mL conical tube placed on ice.</li>
		<li>Centrifuge at 600 x g for 6 min at 4 &#176;C.</li>
		<li>Discard the supernatant. Wash the cell pellet with 5 mL of PBS and centrifuge at 600 x g for 6 min at 4 &#176;C.</li>
		<li>Discard the supernatant, and resuspend the cell pellet in 1 mL of RPMI 10% FBS-20 &#181;g/mL DNase. Keep on ice, protected from light until cell staining.</li>
	</ol>
	</li>
	<li>Single-cell suspension preparation from lung 
	NOTE: Each lung and small intestine should be processed in parallel by two people to avoid extended handling times, which result in low cell viability.
	<ol>
		<li>Prepare 4 mL of lung digestion medium (Table 1) per sample processed.</li>
		<li>Remove the RPMI medium from the well containing the lungs from step 3.2.3. Cut the lung into small pieces with fine scissors.</li>
		<li>Transfer the lung pieces to a 15 mL conical tube with a spatula. Add 4 mL of lung digestion medium (Table 1).</li>
		<li>Incubate for 30 min at 37 &#176;C, with shaking at 250 rpm. When finished, keep on ice until each sample is processed.</li>
		<li>Filter each sample through a 100 &#181;m cell strainer, positioned in a single well of a 6-well culture plate. Dissociate the tissue with a syringe plunger.</li>
		<li>Recover each sample in a 15 mL conical tube, and add 4 mL of R-2 medium (Table 1). Keep on ice while the other samples are processed.</li>
		<li>Centrifuge at 600 x g for 5 min at 4 &#176;C. Discard the supernatant and resuspend the cell pellet in 1 mL of R-5 medium (Table 1).</li>
		<li>While centrifuging, prepare 4 mL of 27.5% density-gradient solution (Table 1) per sample to enrich the hematopoietic cell fraction15,16. This strategy for single-cell separation is more efficient, cost-effective, and less toxic compared to other similar density gradient media17.</li>
		<li>Add 4 mL of 27.5% density-gradient solution to the 1 mL of cell suspension from step 4.10.7, and shake vigorously.</li>
		<li>Slowly add 1 mL of R-5 medium (Table 1) on top of the mixed suspension to create two phases.</li>
		<li>Centrifuge at 1,500 x g for 20 min at RT, with low acceleration and the brake off. Observe the ring formed between the two phases.</li>
		<li>Recover the ring formed between the two phases with a 1 mL micropipette, and resuspend in 4 mL of R-2 medium.</li>
		<li>Centrifuge at 450 x g for 5 min at 4 &#176;C. Discard the supernatant. Resuspend the cell pellet in 1 mL of ACK buffer (Table 1), and incubate for 1 min at RT.</li>
		<li>Add 4 mL of R-5 medium and centrifuge at 450 x g for 5 min at 4 &#176;C. Discard the supernatant and resuspend the cell pellet in 1 mL of R-10 medium. Keep on ice, protected from light until staining for flow cytometry.</li>
	</ol>
	</li>
	<li>Single-cell suspension preparation from lymph nodes
	<ol>
		<li>Place the lymph nodes from steps 3.2.2 or 3.3.3 between two pieces of mesh in a well from a 6-well culture plate, and dissociate with a syringe plunger.</li>
		<li>Recover the cell suspension in a 1.5 mL conical tube and centrifuge at 450 x g for 5 min at 4 &#176;C.</li>
		<li>Discard the supernatant, and resuspend the cell pellet in 1 mL of R-10 medium. Keep on ice, protected from light until staining for flow cytometry. 
		NOTE: If clumps are visible, filter the sample through a 100 &#181;m cell strainer.</li>
	</ol>
	</li>
</ol>
5. Cell staining for flow cytometry (Figure 2 and Figure 3)
NOTE: Centrifuge the lymph node cell suspensions from step 4.11.3 at 450 x g for 5 min at 4 &#176;C, and resuspend the cell pellet in 500 &#181;L of FACS buffer (Table 1).
<ol>
	<li>Cell staining for identification of ILC2s (Figure 3 and Supplementary Figure 2) 
	NOTE: This staining procedure is specific for the identification of IL-9-expressing ILC2 cells.
	<ol>
		<li>Plate 150 &#181;L per lung sample (approximately 1.8 x 106 cells) from step 4.10.14, and 50 &#181;L per lymph node sample from step 4.11.3 (approximately 0.7 x 106 and 2.2 x 106 cells for mediastinal and mesenteric lymph node samples, respectively) in a 96-well conical bottom culture plate. Add 100 &#181;L of FACS buffer, and centrifuge at 450 x g for 5 min at 4 &#176;C.</li>
		<li>Plate 100 &#181;L per small intestine sample (approximately 2.7 x 106 and 0.6 x 106 cells for intraepithelial and lamina propria samples, respectively) from steps 4.8.3 and 4.9.7 in a 96-well conical bottom culture plate. Add 150 &#181;L of FACS buffer and centrifuge at 450 x g for 5 min at 4 &#176;C.</li>
		<li>Discard the supernatant, and resuspend each cell pellet in 50 &#181;L of the biotinylated antibody cocktail (Table 2) diluted in FACS buffer. Incubate for 30 min at 4 &#176;C protected from light. 
		NOTE: Use FACS buffer with 20 &#181;g/mL DNase for intraepithelial and lamina propria samples.</li>
		<li>Add 150 &#181;L of FACS buffer. Centrifuge at 450 x g for 5 min at 4 &#176;C.</li>
		<li>Discard the supernatant, and resuspend the cell pellet in 200 &#181;L of FACS buffer. Centrifuge again and discard the supernatant.</li>
		<li>Resuspend the cell pellet in 50 &#181;L of the antibody/stain cocktail (Table 2), and incubate for 30 min at 4 &#176;C protected from light.</li>
		<li>Add 150 &#181;L of FACS buffer. Centrifuge at 450 x g for 5 min at 4 &#176;C.</li>
		<li>Discard the supernatant, and resuspend the cell pellet in 200 &#181;L of FACS buffer. Centrifuge again and discard the supernatant. Repeat the wash (step 5.1.7), and discard the supernatant.</li>
		<li>Resuspend the cell pellet in 300 &#181;L of FACS buffer, and analyze by flow cytometry.</li>
		<li>For the small intestine and lung samples, use the gating strategy: lymphocytes, single cells, live cells, hematopoietic cells, CD90+Lineage- cells, ST2+ cells, and IL-9+ cells (Figure 3A,B and Supplementary Figure 2A). For the lymph node samples, use the gating strategy: live cells, single cells, CD90+ Lineage- cells, ST2+ cells, and IL-9+ cells (Supplementary Figure 2B,C).</li>
	</ol>
	</li>
	<li>Cell staining for identification of IL-9-producing lymphocytes (Figure 2 and Supplementary Figure 3)
	<ol>
		<li>Transfer 800 &#181;L per lung sample from step 4.10.14 to a 1.5 mL tube (approximately 14.6 x 106 cells). Centrifuge at 450 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
		<li>For lymph node samples, plate 400 &#181;L of the cell suspension from step 4.11.3 (approximately 5.6 x 106 and 17.5 x 106 cells for mediastinal and mesenteric lymph node samples, respectively) in a 96-well conical bottom plate in two steps.
		<ol>
			<li>Transfer 200 &#181;L first, centrifuge at 450 x g for 5 min at 4 &#176;C, and discard the supernatant.</li>
			<li>Transfer another 200 &#181;L to the corresponding well. Centrifuge at 450 x g for 5 min at 4 &#176;C, and discard the supernatant.</li>
		</ol>
		</li>
		<li>Resuspend the cell pellet in 50-400 &#181;L of the antibody/stain cocktail (Table 3), and incubate for 30 min at 4 &#176;C protected from light. 
		NOTE: Lung samples should be resuspended in 400 &#181;L, mediastinal lymph node samples in 50 &#181;L, and mesenteric lymph node samples in 100 &#181;L of the staining cocktail.</li>
		<li>Add 150 &#181;L of FACS buffer to the mediastinal lymph node samples and 100 &#181;L to the mesenteric lymph node samples. Centrifuge at 450 x g for 5 min at 4 &#176;C, and discard the supernatant.</li>
		<li>Wash the lung and lymph node samples by resuspending the pellets in 1 mL and 200 &#181;L of FACS buffer, respectively. Centrifuge at 450 xg for 5 min at 4 &#176;C. Discard the supernatant and repeat the wash.</li>
		<li>Resuspend the lung samples in 600 &#181;L of FACS buffer, and analyze them by flow cytometry. Use the gating strategy: lymphocytes, single cells, live cells, hematopoietic cells, CD4+TCR&#946;+ cells, and IL-9+ cells (Figure 2A).</li>
		<li>Resuspend the lymph node samples in 300 &#181;L of FACS buffer, and analyze by flow cytometry. Use the gating strategy: lymphocytes, single cells, live cells, CD4+ TCR&#946;+ cells, and IL-9+ cells (Figure 2B and Supplementary Figure 3A).</li>
	</ol>
	</li>
</ol>
6. Determination of absolute numbers of cells in single-cell suspensions
<ol>
	<li>Dilute the samples from isolating steps 4.8.3, 4.9.7, 4.10.14, and 4.11.3 with PBS at a 1:20 ratio (10 &#181;L of sample + 190 &#181;L of PBS).</li>
	<li>Mix 10 &#181;L of each diluted sample from step 6.1 with 10 &#181;L of Trypan Blue. Load 10 &#181;L into a hemocytometer and count the live cells, considering each dilution.</li>
	<li>Obtain the absolute numbers of cells by multiplying the percentage of the population of interest from live cells determined by flow cytometry (viability dye negative cells) by the total number of live cells in the single-cell suspension after isolation, and dividing this number by 100 (Supplementary Figure 4, Supplementary Figure 5, and Supplementary Figure 6). 
	Absolute number = (Percentage of the population of interest from live cells determined by flow cytometry x Total number of live cells in the single-cell suspension)/100</li>
</ol>

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The authors have nothing to disclose.

A complete understanding of intestinal parasite-host interactions and immune responses to helminth infection requires the identification and analysis of the different cell populations and effector molecules that are key for the induction of tissue remodeling and worm expulsion. Soil-transmitted helminth infections represent a big problem in developing countries throughout the world. However, until recently, a protocol that allowed for the analysis of rare cell populations present in the small intestine, the main organ af...

Hookworms are intestinal parasites that infect approximately 700 million people worldwide, mostly in tropical areas in underdeveloped countries. High-intensity infections with Ancylostoma duodenale and Necator americanus, the most common hookworm parasites in humans, cause anemia and protein deficiency that can result in delayed growth and mental development1. N. americanus and the rodent parasite Nippostrongylus brasiliensis induce a prototypical type 2 immune response in their host and share similarities in their life cycle. Hence, the infection of mice with N. brasiliensis is the most commonly used model for human hookworm infections. Stage 3 (L3) N. brasiliensis infective larvae move from the skin to the lung in the first few hours post-infection. Once in the lung, they become L4 and migrate up the trachea to get swallowed, pass through the stomach, and reach the gut to become adults (L5) within 4-5 days. In the gut, L5 worms lay eggs that are excreted in the feces to restart the parasite life cycle2.
The immune response induced by N. brasiliensis is characterized by an increase in several type 2 cytokines, including IL-4, IL-5, IL-9, IL-10, and IL-13, along with eosinophilia, basophilia, goblet and mast cell hyperplasia, and heightened IgG1 and IgE production. Most of the studies trying to identify and define the immune responses elicited upon N. brasiliensis infection are centered on the role of IL-4 or IL-13 in this model3. However, the identification and characterization of IL-9-expressing cells and the function of this cytokine had been largely overlooked, until Licona-Lim&#243;n et al. published the first study demonstrating a critical role for IL-9 in the immune response against N. brasiliensis. Using reporter mice, this study described T cells (mostly T helper 9) and type 2 innate lymphoid cells (ILC2s) as the main cellular subsets expressing IL-9 upon infection4.
Isolation and characterization of immune cells from helminth-infected lungs is feasible, and has been extensively reported3,4. However, because of the inherent tissue remodeling and mucus production, to do so in the infected gut proved to be a technical challenge, until the recent publication of Ferrer-Font et al.5. The group outlined a protocol to isolate and analyze single-cell suspensions of immune subsets from Heligmosomoides polygyrus-infected murine intestines. Based on it , we have now standardized a protocol for isolation and cytometric analysis of IL-9-expressing lymphoid cells from the N. brasiliensis infected gut. In addition, we have established IL-9 kinetics from different cellular sources and anatomical locations throughout the infection.
Characterizing the distinct cell populations involved in this infection is vital for a wider understanding of the immune response to the parasite and its interaction with the host. This comprehensive protocol provides a clear route to isolate and analyze IL-9-producing cells from desired organs at disease stages of interest, allowing for a sharp improvement of the knowledge about the role of these cells in N. brasiliensis infection and parasite infections in general.

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a strategy for the study of il-9-producing lymphoid cells in the nippostrongylus brasiliensis infection model

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response against helminth infections, where it plays an important role in the expulsion of the parasite. In a murine model of Nippostrongylus brasiliensis infection, IL-9 is produced mainly by CD4+ T lymphocytes and innate lymphoid cells found in the lung, small intestine, and draining lymph nodes. Given the technical difficulties involved in the intracellular staining of IL-9, as well as the complexity of isolating hematopoietic cells from the small intestine upon infection, there is a pressing need for a comprehensive but straightforward protocol to analyze the expression of IL-9 in different lymphoid and non-lymphoid tissues in this model. The protocol described here outlines the kinetics of IL-9 produced by CD4+ T cells and innate lymphoid cells in the lung and small intestine, the main organs targeted by N. brasiliensis, as well as in the mediastinal and mesenteric lymph nodes, throughout the infection. In addition, it details the number of larvae needed for infection, depending on the cell type and organ of interest. This protocol aims to assist in the standardization of assays to save time and resources by offering the opportunity to focus on the specific cells, organs, and disease stages of interest in the N. brasiliensis infection model.

Mice were subcutaneously injected with 200 L3 stage N. brasiliensis larvae, or with PBS for sham controls. The number of larvae used in this protocol was adjusted in order to isolate viable cells from the lungs, lymphoid tissue, and the small intestine, unlike previous reports where higher loads of worms were used to detect cells in lymphoid tissues and lungs only4. Lungs, mediastinal lymph nodes, mesenteric lymph nodes, and the small intestine were harvested at days 0, 4, 7, and 10 post-...

Watch this Scientific Journal Video about A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model at JoVE.com

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9-expressing T and ILC2 cells are induced during N. brasiliensis infection, yet their characterization has been largely overlooked in the infected intestine due to their low frequency and differential kinetics. This protocol describes the isolation of these cells from different target organs and confirmation of their identity via flow cytometry at different infection stages.

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response ...

This protocol allows for the isolation and characterization of lymphoid cells expressing IL-9 in N.brasiliensis non-lymphoid infected tissues including the infected cut which recently was technically invisible.This protocol also provides an alternative to analyze populations of cells from peripheral tissues previously unappreciated in infection due to the intrinsic difficulties of isolating cells from these anatomical sites.This method allows the study of difficult-to-isolate populations from N.brasiliensis infected cut and could be applied to other helminthic infections, broadening our understanding of hookworm infections in humans.Demonstrating the procedure will be Ofelia Mu

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Immunology and Infection

1.2K 조회수.  Universidad Nacional Autónoma de México. This protocol allows for the isolation and characterization of lymphoid cells expressing IL-9 in N.brasiliensis non-lymphoid infected tissues including the infected cut which recently was technically invisible.This protocol also provides an alternative to analyze populations of cells from peripheral tissues previously unappreciated in infection due to the intrinsic difficulties of isolating cells from these anatomical sites.This method allows the study of difficult-to-isolate populations from...

비디오: A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...