Name: detecting migration and infiltration of neutrophils in mice
Uploaded: 2020-02-06T13:30:00.000+00:00
Duration: 10 min 33 s
Description: Watch this Scientific Journal Video about Detecting Migration and Infiltration of Neutrophils in Mice at JoVE.com

This work was supported by the National Natural Science Foundation of China (grant numbers 81430099 and 31500704), International Cooperation and Exchange Projects (grant number 2014DFA32950), and the research program of Beijing University of Chinese Medicine (grant numbers BUCM-2019-JCRC006 and 2019-JYB-TD013).

All experimental procedures were reviewed and approved by the Beijing University of Chinese Medicine Animal Care and Use Committee.
NOTE: C57BL/6 mice (7-8 weeks old) were used.
1. Neutrophil isolation
<ol>
	<li>Acquisition of peritoneal exudate cells
	<ol>
		<li>Prepare fresh 10% proteose peptone solution in ddH2O. Calculate the volume needed according to the number of mice. 
		NOTE: Set the number of mice as N, (2N+1) mL of solution must be dissolved and filtered in advance.</li>
		<li>Spray the workspace with 70% ethanol. Use an insulin injector to draw 1 mL of peptone solution and discharge bubbles.</li>
		<li>Conduct the first intraperitoneal injection of 1 mL of peptone solution per mouse.
		<ol>
			<li>Grab the mouse in a head-down position with one hand. Disinfect the injection spot with alcohol-soaked cotton balls. 
			NOTE: The preferred injection position lies in the lateral aspect of the lower left or right quadrant of the abdomen.</li>
			<li>Infuse the reagents rapidly. Inject 1 mL into the peritoneal cavity of each mouse with the insulin injector. 
			NOTE: The angle between the needle and the skin should be approximately 15&#8722;30&#176; to avoid injuring the intestine or other organs.</li>
		</ol>
		</li>
		<li>Allow inflammatory response to develop overnight. After 12 h, conduct the second injection in the same manner as the first injection.</li>
		<li>Three hours after the second injection, fully anesthetize mice with 5% isoflurane for 5 min in a gas anesthesia chamber at a speed of 2 L/min. Remove the anesthetized mice from the chamber and sacrifice them by cervical dislocation. 
NOTE: Sufficient depth of anesthesia is ensured by pinching the toes before moving mice from the chamber to the single breathing unit. The legs should not move when the toes are pinched. 		
NOTE: All the following steps should be processed in a tissue culture hood.</li>
		<li>Spray the mouse with 70% ethanol. Lay the mouse on a sterile plastic pad and fix the limbs with needles.</li>
		<li>Use a sterile set of surgical tools to make a horizontal incision (~1 cm) in the middle of the lower abdomen. Lift the skin of the upper abdomen with forceps and cut along the midline of the abdomen and expose the intact peritoneal wall.</li>
		<li>Inject 5 mL of sterile RPMI-1640 complete medium into the abdominal cavity with a 30 G x 1/2&#34; needle. Insert the needle through the peritoneal wall with the beveled edge of the needle facing up and inject the entire volume.</li>
		<li>Shake the pad horizontally for 5 min. Massage the abdomen gently several times.</li>
		<li>Inject a 23 G x 1 1/4&#34; needle into the lateral space of the abdomen. Extract the abdominal liquid (~5 mL) and collect it in a 50 mL centrifuge tube. 
		NOTE: Place the tubes on ice as soon as possible in case of neutrophil activation.</li>
		<li>Inject another 5 mL of complete medium and repeat the procedure to remove the remaining cells from the peritoneum. Pool the peritoneal fluid in the 50 mL centrifuge tube.</li>
		<li>Centrifuge the pooled peritoneal fluid at 400 x g for 10 min at room temperature (RT).</li>
		<li>Discard the supernatant. Resuspend the cells in 1 mL of RPMI-1640 complete medium. 
		NOTE: Do not vortex to avoid neutrophil activation.</li>
	</ol>
	</li>
	<li>Isolation of neutrophils
	<ol>
		<li>Add 4 mL of freshly prepared 70.2% density gradient medium (e.g., Percoll) in a 15 mL centrifuge tube.</li>
		<li>Carefully overlay 4 mL of freshly prepared 54.8% density gradient medium on the 70.2% density gradient medium slowly along the edge of the tube with sharp pipette tips in the 15 mL centrifuge tube. 
		NOTE: Exercise caution to avoid disturbing the interface between the 54.8% density gradient medium and 70.2% density gradient medium.</li>
		<li>Carefully overlay the 1 mL peritoneal cell suspension on top of the 54.8% density gradient medium layer slowly with sharp pipette tips (Figure 1A). 
		NOTE: Exercise caution to avoid disturbing the interface between the cell suspension and 54.8% density gradient medium.</li>
		<li>Centrifuge at 1,500 x g for 30 min at 22 &#176;C without braking.</li>
		<li>Collect the neutrophils at the interface of the 54.8% density gradient medium and 70.2% density gradient medium layers (Figure 1B) to a new tube.</li>
		<li>Add 1 mL of RPMI-1640 complete medium to the collected cells and carefully resuspend cells by gently pipetting several times. Centrifuge at 100 x g for 10 min at RT and carefully remove the supernatant.</li>
		<li>Repeat the wash step (step 1.2.6) once.</li>
		<li>Add 0.5 mL of culture medium to the pellet and resuspend cells by gently pipetting several times. Take a 50 &#181;L aliquot to count the cells using an automatic hematology analyzer.</li>
	</ol>
	</li>
</ol>
2. Neutrophil migration assay
<ol>
	<li>Measure neutrophil migration by Transwell assay9 or Zigmond chamber assay as previously described10,11.</li>
</ol>
3. Air pouch assay
<ol>
	<li>First air injection
	<ol>
		<li>On day 0, fully anesthetize mice with 5% isoflurane for 3 min in a gas anesthesia chamber at a speed of 2 L/min, and maintain the anesthesia of each mouse in a single breathing unit with 2% isoflurane at a speed of 0.5 L/min. 
NOTE: Sufficient depth of anesthesia is ensured by pinching the toes before moving mice from the chamber to the single breathing unit. The legs should not move when the toes are pinched.</li>
		<li>Use a 0.22 &#181;m filter attached to a 5 mL syringe to obtain a 3 mL volume of sterilized air.</li>
		<li>Lift the back skin of the anesthetized mouse with tweezers and subcutaneously inject 3 mL of sterilized air using a 26 G x 3/8&#34; needle.</li>
		<li>After treatment, remove the mice from the breathing unit. Monitor the mice to ensure they are alive until they start to move around.</li>
	</ol>
	</li>
	<li>Second air injection
	<ol>
		<li>On day 3, inject an additional 3 mL of sterilized air into the previously established air pocket to sustain the air pouch as described in section 3.1.</li>
	</ol>
	</li>
	<li>Treatment
	<ol>
		<li>On day 6, 6 h before sacrifice, inject different treatments into the air pouch. Inject 1 mL of phosphate-buffered saline (PBS) as a negative control. Inject 1 mL of 1 &#181;g/mL LPS as the positive control to induce local inflammation.</li>
		<li>Fully anesthetize mice with 5% isoflurane for 3 min in a gas anesthesia chamber at a speed of 2 L/min, and maintain the anesthesia of each mouse in a single breathing unit with 2% isoflurane at a speed of 0.5 L/min. Prepare wash buffer according to Table of Materials. 
NOTE: Sufficient depth of anesthesia is ensured by pinching the toes before moving mice from the chamber to the single breathing unit. The legs should not move when the toes are pinched.</li>
		<li>For each air pouch, wash the air pouch with 1 mL of wash buffer and collect the inflammatory exudate in a 15 mL centrifuge tube. Wash the air pouch with 2 mL of wash buffer 2x and collect the inflammatory exudate in the same centrifuge tube.</li>
		<li>Centrifuge at 100 x g for 10 min at RT. Discard the supernatant and resuspend cells in 1 mL of wash buffer. Count the cells to quantify the neutrophil ratio using the automatic hematology analyzer. 
		NOTE: See representative results in Figure 2.</li>
	</ol>
	</li>
</ol>
4. Induction of the adjuvant-induced arthritis (AA) mouse model
<ol>
	<li>Suspend complete Freund&#39;s adjuvant (CFA) by vortexing at least 5 s, then draw 100 &#181;L of suspension into an insulin injector. 
NOTE: We recommend using completely new CFA in experiments to ensure sterility.</li>
	<li>Anesthetize mice as described in step 3.1.1.</li>
	<li>Mark the chosen paw and inject 20 &#181;L of CFA into the ankle joint space. Inject 20 &#181;L of suspension into four periarticular spots on the chosen paw (80 &#181;L in total).</li>
	<li>Remove mice from the breathing unit and put the processed mice in a new chamber. Monitor mice to ensure that they are breathing until they regain the ability to move.</li>
	<li>Every 3 days, assess the joint diameter by measuring the ankle joint diameter using a pocket thickness gauge (Figure 3A).</li>
	<li>Every 3 days, assess arthritis severity by arthritis scoring criterion (Figure 3C): 0, normal, no evidence of erythema and swelling; 1, the mildest arthritis, erythema and mild swelling confined to the tarsals or ankle joint; 2, moderate arthritis, erythema and mild swelling extending from the ankle to the tarsals; 3, severe arthritis, erythema and moderate swelling extending from the ankle to metatarsal joints; 4, the most severe arthritis, erythema and severe swelling encompass the ankle, foot and digits, or ankylosis of the limb.</li>
</ol>
5. Immunohistochemical staining of joint sections
<ol>
	<li>Joint isolation
	<ol>
		<li>Sacrifice the mouse in section 4 using cervical dislocation after anesthesia with isoflurane. Spray the mouse with 70% ethanol.</li>
		<li>Remove the skin and part of the muscle from the hind leg with tweezers and scissors. Spray the joint with 70% ethanol and remove the rest of the muscles using a paper towel.</li>
		<li>Fix the ankle joint in 4% paraformaldehyde for 2 days at RT. Decalcify the joint in 10% EDTA for 1 month at RT and change the medium weekly.</li>
		<li>Embed the tissue in paraffin and prepare 4-&#181;m-thick tissue sections.
		<ol>
			<li>Place tissue in a marked mold with certain volume of liquid paraffin. Cool briefly.</li>
			<li>Set the thickness at 4 &#181;m and cut slices on a microtome. Float sections in a 43 &#176;C water bath.</li>
			<li>Mount the sections onto slides and put the slides into the oven at 70 &#176;C for 2 h. For future use, conserve the slides at -20 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Safranin O and fast green staining of joint sections 
	NOTE: The following staining steps are conducted at RT.
	<ol>
		<li>Place the slides from step 5.1.4.3 in a rack and perform the following washes to rehydrate at RT: xylene for 5 min (3x), 100% ethanol for 2 min (2x), 95% ethanol for 2 min (2x), 70% ethanol for 2 min, and 50% ethanol for 15 min. Wash in running tap water for 3 min.</li>
		<li>Stain in 0.1% fast green solution for 5 min. Rinse in 1% acetic acid for 10 s.</li>
		<li>Stain in 0.1% safranin O staining solution for 20 min. Immerse the slides in the following washes: 95% ethanol for 2 min (2x), 100% ethanol for 2 min (2x), and xylene for 2 min (2x).</li>
		<li>Mount the tissue sections and observe the tissues under a microscope. 
		NOTE: See representative images in Figure 4A.</li>
	</ol>
	</li>
	<li>Immunohistochemical staining to visualize neutrophils
	<ol>
		<li>Bake the paraffin sections for 2 h at 78 &#176;C. Place the slides in a rack and perform the following washes to rehydrate at RT: xylene for 15 min (2x), 100% ethanol for 5 min (2x), 95% ethanol for 5 min, 80% ethanol for 5 min, H2O for 3 min, and PBS for 3 min. 
		NOTE: Do not let slides dry at any time during this step.</li>
		<li>Add one drop of permeabilization buffer to cover the tissue. Incubate sections in a humidity-controlled tray at 37 &#176;C.</li>
		<li>Rinse slides in PBS for 3 min (3x). Avoid rinsing the tissue directly.</li>
		<li>Perform heat-induced antigen epitope retrieval using a pressure boiler.
		<ol>
			<li>Arrange slides in a rack. Immerse slides in the pressure boiler filled with retrieval buffer.</li>
			<li>Put the pressure boiler on a microwave oven. Set the microwave oven at 600 W and heat the slides for 10 min.</li>
			<li>After boiling, keep slides in the boiler to cool to 90 &#176;C. Take out the slides and rinse them in PBS for 3 min (3x).</li>
		</ol>
		</li>
		<li>Quench endogenous peroxidase activity in freshly prepared 3% H2O2 at RT for 15 min. Rinse slides in PBS for 3 min (3x).</li>
		<li>Outline a large circle around the sample with a hydrophobic pen, avoid touching the sample. Block with 3% bovine serum albumin (BSA) in a humidity-controlled chamber at 37 &#176;C for 60 min.</li>
		<li>Remove blocking solution. Add 50 &#181;L of PBS-diluted primary antibody to each section quickly. Then, incubate the slides in a humidity-controlled tray at 4 &#176;C overnight. 
		NOTE: Different dilution ratios are used for different antibodies (1:25 for MPO and 1:20 for NE).</li>
		<li>On the second day, take out the tray and let it stand at RT for 30 min. Then, rinse the slides in PBS for 3 min (3x).</li>
		<li>Add 50 &#181;L of PBS-diluted secondary antibodies to the tissue. 
		NOTE: Different dilution ratios were applied: 1:1,000 for MPO and 1:1,500 for NE.</li>
		<li>Incubate slides in a humidity-controlled tray at 37 &#176;C for 30 min. Then, rinse the slides in PBS for 3 min (3x).</li>
		<li>Develop in diluted 3,3&#39;-diaminobenzidine (DAB) solution for 5 min. Keep an eye on the reaction in case of the development of a dark color. Rinse the slides in distilled water.</li>
		<li>Counterstain the slides in hematoxylin for 10 s. Rinse the slides in tap water for 5 min.</li>
		<li>Rinse in acid alcohol superfast differentiation solution for 3 s. Then rinse in tap water for 10 min.</li>
		<li>Immerse the slides in the following washes at RT: 80% ethanol for 5 min, 95% ethanol for 5 min, 100% ethanol for 5 min, and xylene for 15 min (2x). Fix the coverslip with mounting solution. Observe the tissue under a microscope. 
		NOTE: See representative images in Figure 4B.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Detailed protocols of highly-purified neutrophils from peripheral blood7, bone marrow and tissues18 have been available for a long time. Here we adopt a method of isolating neutrophils from peritoneal fluid19 in which mature neutrophils remain inactivated for further anti-inflammatory and antioxidant studies.
We used the air pouch experiment to explore the LPS-induced infiltration of neutrophils in vivo. This method has been proposed as a potential method for directly measuring cell infiltration in the general inflammatory environment in vivo.
It is noteworthy that the air pouch assay only demonstrates the neutrophil&#39;s function in an organ-nonspecific manner and removes several steps of the leukocyte recruitment cascade. Since neutrophil recruitment to specific organs can rely on different organ properties, adhesion molecules, and chemokines, exploring the neutrophil functional state in organ-specific conditions is of great importance for studying the role that neutrophils potentially play in certain diseases3. It has been suggested that endpoint models are required to investigate neutrophil infiltration. Therefore, performing immunohistochemical staining on the joint sections of mice in the AA model can provide an insightful perspective on the neutrophils in the joint space. According to our data, vast numbers of neutrophils are recruited into the joint tissue and serve as fundamental evidence for further research on interrupting the infiltration of neutrophils to treat RA. In addition, comprehensive assays, for example, safranin O-fast green staining, are required to evaluate the disease model for further study.
Neutrophils are the major subset of infiltrating inflammatory cells and work as the first line of defense against invading pathogens or tissue injury20,21. If neutrophils infiltrate tissues in large numbers, high levels of cytokines and NETs are secreted, which together may overwhelm the protective mechanisms in tissues and lead to tissue damage. Tissue injury further stimulates neutrophil infiltration, thus forming a vicious cycle22. Interfering with cell migration by means of trapping activated cells in lymphoid organs has been proposed as an important therapeutic approach and has been applied in clinical trials with various side effects23. Discovering a novel treatment to concurrently regulate neutrophil migration and inflammatory activity is a promising strategy for treating inflammatory diseases in the future. Furthermore, if motility-regulating agents are combined, neutrophil-mediated drug delivery24 can increase drug specificity, thus decreasing side effects.
However, further modifications can be combined to broaden the application of the above-mentioned assays. For example, in the AA mouse model, to obtain the overall impression of infiltration of inflammatory cells in the joint, researchers are encouraged to enzymatically digest joints to release the resident leukocyte populations and then apply flow cytometry to count the populations.
The protocols herein include three ways to assess neutrophil migration and infiltration. The application of these protocols is useful for discovering potential treatments for RA and other inflammatory diseases involving neutrophils.

Neutrophils are the most abundant white blood cell and account for 50&#8722;70% of the whole white blood cell population in humans1. Neutrophils are one of the primary responders during acute inflammation. Neutrophils can be recruited to inflammation sites via the guidance of cytokines and chemokines released by tissue-resident cells2,3,4, which is mediated by the interactions between cell adhesion molecules on the surface of neutrophils and vascular endothelium cells5. Neutrophils are fundamental to host defense and play a role in pathologic inflammatory reactions due to their powerful capacity to damage tissue via the release of reactive oxygen species (ROS) and other tissue-damaging molecules3,6.
Previous studies have described several neutrophil isolation protocols from mice or humans. Oh et al. demonstrated a density gradient separation method to isolate human neutrophils from whole human blood7. However, the isolation of sufficient neutrophils from mouse blood is difficult because of the small blood volume. Alternatively, large numbers of pure and viable mouse neutrophils can be elicited from mouse peritoneal fluid, and these purified neutrophils can be used ex vivo to examine several aspects of cellular functions ex vivo, including neutrophil infiltration, migration, chemotaxis, oxidative burst, cytokine and neutrophil extracellular trap (NET) production8. Transwell assays9 or Zigmond chamber assays10,11 can be used to evaluate neutrophil migration in vitro. The air pouch model is used to evaluate the migration and infiltration of neutrophils in vivo. The subcutaneous air pouch model is a convenient in vivo animal model to study the migration of inflammatory cells.
Traditionally, neutrophils were considered as pathogen eliminators in acute phases of inflammation. However, recent findings have shown that neutrophils are complicated cells that perform a significant variety of specialized functions. Neutrophils can regulate many processes such as acute injury and repair, tumorigenesis, autoimmune response, and chronic inflammation12,13. Neutrophils also modulate adaptive immune responses and can regulate B cells and T cells14,15. Substantial shortage of neutrophils leads to mortality or severe immunodeficiency in humans and neutrophil depletion in mice leads to fatality, while excessive activation or recruitment of neutrophils in organs causes several immune diseases, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE)6. Neutrophils are the most abundant cells in the synovial fluid of RA patients. Neutrophils produce excessive amounts of myeloperoxidase (MPO) and neutrophil elastase (NE) via degradation, which exacerbates cartilage erosion. MPO is a peroxidase enzyme mainly expressed in the granules of neutrophils16. NE is associated with articular cartilage destruction17. MPO and NE could be used to evaluate the status of neutrophil migration and infiltration in the tissue of RA patients.
This article provides three conventional methods to evaluate the migration of normal neutrophils induced both in vivo and in vitro, as well as the infiltration of pathological neutrophils in a mouse joint-specific inflammation model.

<table><tbody><tr><td>0.1% Fast Green Solution</td><td>Solarbio</td><td>8348b</td><td>Transfer 20 mg of fast grene FCF in one vial into another 100 mL beaker. Add 20 mL of H&lt;sub&gt;2&lt;/sub&gt;O into the beaker and dissolve the stain by stirring to make 0.1% fast green solution, and filter it using a Nalgene PES 75mm filter</td></tr><tr><td>0.1% Safranin O Staining Solution</td><td>Solarbio</td><td>8348a</td><td>Transfer 20 mg of safranin O stain in one vial into a 100 ml beaker. Add 20 mL of H&lt;sub&gt;2&lt;/sub&gt;O into the beaker and dissolve the stain by stirring to make 0.1% safranin O staining solution, and filter it using a Nalgene PES 75mm filter</td></tr><tr><td>0.5 M Ethylenediaminetetraacetic acid solution (EDTA), pH 8.0</td><td>Sigma</td><td>324506</td><td>Sterile</td></tr><tr><td>100% Ethanol</td><td>Beijing Chemical Works</td><td></td><td></td></tr><tr><td>100% Methanol</td><td>Beijing Chemical Works</td><td></td><td></td></tr><tr><td>15 mL Conical Polypropylene Centrifuge Tube</td><td>Falcon</td><td>14-959-53A</td><td></td></tr><tr><td>23 G x 1 1/4&quot; Needle</td><td>BD</td><td>305120</td><td></td></tr><tr><td>26 G x 3/8&quot; Needle</td><td>BD</td><td>305110</td><td></td></tr><tr><td>3% bovine serum albumin (BSA)</td><td></td><td></td><td>Dissolve 0.3 g BSA in 10 mL PBS</td></tr><tr><td>3% H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>Mix 1 mL 30% H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; with methanol with 9 mL methanol</td></tr><tr><td>3,3&#039;-diaminobenzidine (DAB) kit</td><td>ZSGB-BIO</td><td>ZLI-9018</td><td></td></tr><tr><td>30 G x 1/2&quot; Needle</td><td>BD</td><td>305106</td><td></td></tr><tr><td>30% H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Beijing Chemical Works</td><td></td><td></td></tr><tr><td>5 mL Syringe</td><td>BD</td><td>Z683574</td><td></td></tr><tr><td>50 mL Conical Polypropylene Centrifuge Tube</td><td>Falcon</td><td>14-432-22</td><td></td></tr><tr><td>50% Ethanol</td><td></td><td></td><td>Mix 500 mL 100% ethanol with 500 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>54.8% Percoll</td><td></td><td></td><td>Mix 2.74 mL SIP with 2.26 mL 1&amp;times;PBS, stand still</td></tr><tr><td>70% Ethanol</td><td></td><td></td><td>Mix 700 mL 100% ethanol with 300 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>70.2% Percoll</td><td></td><td></td><td>Mix 3.51 mL SIP with 1.49 mL 1&amp;times;PBS, stand still</td></tr><tr><td>80% Ethanol</td><td></td><td></td><td>Mix 800 mL 100% ethanol with 200 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>95% Ethanol</td><td></td><td></td><td>Mix 950 mL 100% ethanol with 50 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Acid Alcohol Superfast Differentiation Solution</td><td>Beyotime</td><td>C0165S</td><td></td></tr><tr><td>ANTIBODIES</td><td></td><td></td><td></td></tr><tr><td>Anti-Myeloperoxidase Antibody</td><td>Abcam</td><td>ab208670</td><td></td></tr><tr><td>Anti-Neutrophil Elastase Antibody</td><td>Abcam</td><td>ab21595</td><td></td></tr><tr><td>Automatic Hematology Analyzer</td><td>Sysmex</td><td>XS-800i</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>VWR</td><td>0332-100G</td><td></td></tr><tr><td>Complete Freund&#039;s Adjuvant, 10 mg/ml</td><td>sigma</td><td>1002036152</td><td></td></tr><tr><td>Cover Slip</td><td>CITOGLAS</td><td>10212432C</td><td></td></tr><tr><td>Dial Thickness Gauge</td><td>Mitutoyo</td><td>7301</td><td></td></tr><tr><td>Eppendorf Microtubes, 1.5 mL</td><td>Sigma</td><td>Z606340</td><td></td></tr><tr><td>Foetal Bovine Serum (FBS) Premium</td><td>PAN</td><td>P30-1302</td><td></td></tr><tr><td>Gas Anesthesia System</td><td>ZS Dichuang</td><td>ZS-MV-IV</td><td></td></tr><tr><td>Goat Anti-Rabbit IgG H&amp;amp;L (HRP)</td><td>PPLYGEN</td><td>C1309</td><td>This is the secondary antibody used in the immunohistochemical staining.</td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Biological Industries, Beth HaEmek, Israel</td><td>02-016-1A</td><td>Sterile</td></tr><tr><td>Hematoxylin Staining Solution</td><td>ZSGB-BIO</td><td>ZLI-9609</td><td></td></tr><tr><td>Lipopolysaccharide (LPS)</td><td>Sigma</td><td>L3012</td><td></td></tr><tr><td>MEDIA AND SUPPLEMENTS</td><td></td><td></td><td></td></tr><tr><td>Modified Safranin O-fast Green FCF Cartilage Stain Kit</td><td>Solarbio</td><td>G1371</td><td></td></tr><tr><td>N-formyl-Met-Leu-Phe (fMLP)</td><td>Sigma</td><td>47729</td><td></td></tr><tr><td>Penicillin Streptomycin Solution, 100&amp;times;</td><td>Invitrogen</td><td>1514022</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>10245207</td><td>Density gradient medium</td></tr><tr><td>Permeabilization Buffer</td><td></td><td></td><td>Mix 100 &amp;mu;L Triton X-100 with 1 L dH&lt;sub&gt;2&lt;/sub&gt;O to get 0.01% Triton X-100</td></tr><tr><td>Phosphate Buffer Saline (PBS), 1&amp;times;</td><td></td><td></td><td>Mix 90% ddH&lt;sub&gt;2&lt;/sub&gt;O with 10% (v/v) 10&amp;times;PBS, autoclaved</td></tr><tr><td>Phosphate Buffer Saline (PBS), 10&amp;times;</td><td></td><td></td><td>Dissolve 16 g NaCl, 0.4 g KCl, 2.88 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&amp;middot;&lt;/sub&gt;2H&lt;sub&gt;2&lt;/sub&gt;O, 0.48 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (anhydrous) in 200 mL ddH&lt;sub&gt;2&lt;/sub&gt;O, adjust pH 7.4, autoclaved</td></tr><tr><td>PLASTIC WARES AND EQUIPMENTS</td><td></td><td></td><td></td></tr><tr><td>POWDER</td><td></td><td></td><td></td></tr><tr><td>Proteose Peptone</td><td>Oxoid</td><td>1865317</td><td></td></tr><tr><td>Retrieval Buffer</td><td></td><td></td><td>Mix 18 mL retrieval buffer A with 82 mL retrieval buffer B, add dH&lt;sub&gt;2&lt;/sub&gt;O to 1000 mL, adjust pH to 6.0</td></tr><tr><td>Retrieval Buffer A Stock for IHC</td><td></td><td></td><td>Dissolve 4.2 g citric acid (C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;7&amp;middot;&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;O) in 200 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Retrieval Buffer B Stock for IHC</td><td></td><td></td><td>Dissolve 5.88 g trisodium citrate dihydrate (C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;Na&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;0) in 200 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Roswell Park Memorial Institute (RPMI)-1640 medium</td><td>Sigma</td><td>R8758</td><td></td></tr><tr><td>RPMI-1640 Complete Medium</td><td></td><td></td><td>RPMI-1640 medium is supplemented with 10% FBS and 1% penicillin/streptomycin.</td></tr><tr><td>Shu Rui U40 Disposable Sterile Insulin Injection Needle 1 mL</td><td>BD</td><td>328421</td><td></td></tr><tr><td>Slide</td><td>CITOGLAS</td><td>10127105P-G</td><td></td></tr><tr><td>SOLUTION</td><td></td><td></td><td></td></tr><tr><td>Stock Isotonic Percoll (SIP)</td><td></td><td></td><td>Mix 90% (v/v) of percoll with 10% (v/v) 10&amp;times;PBS, stand still for 20 min</td></tr><tr><td>Wash Buffer in Air Pouch Assay</td><td></td><td></td><td>Dilute 0.5M EDTA to 10mM with HBSS</td></tr><tr><td>Xylene</td><td>Beijing Chemical Works</td><td></td><td></td></tr></tbody></table>

detecting migration and infiltration of neutrophils in mice

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. Neutrophils can be recruited to inflammation sites via the guidance of cytokines and chemokines. Overwhelming infiltration of neutrophils can lead to indiscriminate tissue damage, such as in rheumatoid arthritis (RA). Neutrophils isolated from peritoneal exudate respond to a defined chemoattractant, N-formyl-Met-Leu-Phe (fMLP), in vitro in Transwell or Zigmond chamber assays. The air pouch experiment can be used to evaluate the chemotaxis of neutrophils towards lipopolysaccharide (LPS) in vivo. The adjuvant-induced arthritis (AA) mouse model is frequently used in RA research, and immunohistochemical staining of joint sections with anti-myeloperoxidase (MPO) or anti-neutrophil elastase (NE) antibodies is a well-established method to measure neutrophil infiltration. These methods can be used to discover promising therapies targeting neutrophil migration.

Peritoneal exudate cells were collected from lavage fluid of mice. Cells were resuspended in 1 mL of RPMI-1640 complete medium, layered onto a two-step (54.8%/70.2%) discontinuous density gradient (Figure 1A), and centrifuged at 1,500 x g for 30 min. Neutrophils (&#8805;95%, ~1 x 107 neutrophils/mouse) were recovered from the lower interface (Figure 1B).
Air pouch experiments were performed to investigate the neutrophil recruitment stimulated by LPS in vivo (Figure 2A). The leukocyte subsets in the air pouch exudates were measured (Figure 2B).
Neutrophil migration in RA was evaluated via the CFA-induced arthritis murine model. Compared with the control group, the AA group showed significant edema in the paw. In the AA group, the ankle joint diameter increased (Figure 3B) and the arthritis score rose consistently (Figure 3D).
Cartilage damage is the representative syndrome of RA, safranin O-fast green cartilage staining was performed to assess the cartilage damage in AA mouse. As shown in Figure 4A, CFA challenge induced a large amount of leukocyte infiltration, significant cartilage erosion and synovial hyperplasia. MPO and NE expression levels are representative markers of neutrophil infiltration. Immunohistochemical assays were performed to observe neutrophil infiltration in joints. MPO and NE expression was significantly upregulated in the joint section (Figure 4B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60543/60543fig1a.jpg" /> 
Figure 1: Neutrophil isolation. (A) Peritoneal exudate cells resuspended in 1 mL of RPMI-1640 complete medium were layered onto a two-step (54.8%/70.2%) discontinuous density gradient. (B) After centrifugation at 1,500 x g for 30 min, neutrophils (&#8805;95%, ~1 x 107 neutrophils/mouse) were recovered from the lower interface. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60543/60543fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60543/60543fig2.jpg" /> 
Figure 2: Representative results of the air pouch assay. (A) Illustration of the air pouch assay. (B) Representative results of leukocyte subset infiltration in the air pouch assay. PBS: control; LPS: 1 &#181;g/mL LPS. Data are presented as the mean &#177; SD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60543/60543fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60543/60543fig3.jpg" /> 
Figure 3: Representative results of the adjuvant-induced arthritis (AA) mouse model. (A) The ankle joint diameter was measured using a pocket thickness gauge. (B) Joint swelling was assessed based on the ankle joint diameter (n = 7). (C) Pictures of each arthritis score. (D) The severity of arthritis was graded on a scale of 0&#8722;4 points (n = 7). Data are presented as the mean &#177; SD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60543/60543fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60543/60543fig4a.jpg" /> 
Figure 4: Representative results of safranin O-fast green cartilage staining and immunohistochemical assay of joint sections from control and AA mice. (A) Representative results of safranin O-fast green cartilage staining of joint sections. (B) Representative results of the expression level of MPO and NE in the ankle joints. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60543/60543fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detecting Migration and Infiltration of Neutrophils in Mice at JoVE.com

Detecting Migration and Infiltration of Neutrophils in Mice

Here, we present three methods to assess neutrophil migration and infiltration both in vivo and in vitro. These methods can be used to discover promising therapeutics targeting neutrophil migration.

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. ...

detecting-migration-and-infiltration-of-neutrophils-in-mice

Nanyang Technological University

Research

JoVE Journal

Biology

11.8K Views.  Beijing University of Chinese Medicine. Deze methode probeert drie veelgebruikte methoden te beschrijven voor het evalueren van de migratie van normale neutrofielen in vivo en in vitro, evenals de infiltratie van pathologische neutrofielen in het model van muisartritis. Wij denken dat deze methode nuttig kan zijn voor andere onderzoekers op dit gebied. Dit protocol bevat de methodologische details om de migratiecapaciteit van neutrofielen in de ontstekingsplaatsen te evalueren met behulp van luchtzaktest en adjuvante-geïnduceerde ...

Video: Detecting Migration and Infiltration of Neutrophils in Mice

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Immunology and Infection

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.
We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.
IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high concentrations in specific and tertiary granules of neutrophils. This protocol details how MUB40&#160;conjugated directly to a fluorophore can be used to stain neutrophils in fixed/permeabilized tissues as well as how this can be used in live-cell imaging to assay for neutrophil activation and de-granulation. Neutrophil detection methods are limited to species-specific monoclonal antibodies, which are not always suitable for certain applications. MUB40 does not penetrate the cell membrane and is thus excluded from lactoferrin stored in non-activated/non-permeabilized neutrophils. MUB40 has the added benefit of recognizing lactoferrin from a broad host range, making it especially useful for comparing results in studies involving multiple research models, reducing the number of duplicate reagents, and simplifying protocols through single-step staining.

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Here, we present a protocol to detect the presence of neutrophils in fixed/permeabilized histology sections and assess the activation state of live purified neutrophils. In particular, the MUB40&#160;peptide binds lactoferrin present in neutrophil-specific and tertiary granules. Exposure of the granule contents through either permeabilization or neutrophil activation allows for the marking of neutrophils.

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high ...

Imaging Neutrophil Migration in the Mouse Skin to Investigate Subcellular Membrane Remodeling Under Physiological Conditions

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first immune cells to reach the site of inflammation and to participate in the innate immune response during infection or tissue damage. So far, neutrophil migration has been successfully visualized using various in vitro experimental systems based on uniform stimulation, or confined migration under agarose, or micro-fluidic channels. However, these models do not recapitulate the complex microenvironment that neutrophils encounter in vivo. The development of multiphoton microscopy (MPM)-based techniques, such as intravital subcellular microscopy (ISMic), offer a unique tool to visualize and investigate neutrophil dynamics at subcellular resolutions under physiological conditions. In particular, the ear of a live anesthetized mouse provides an experimental advantage to follow neutrophil interstitial migration in real-time due to its ease of accessibility and lack of surgical exposure. ISMic provides the optical resolution, speed, and depth of acquisition necessary to track both cellular and, more importantly, subcellular processes in 3D over time (4D). Moreover, multi-modal imaging of the interstitial microenvironment (i.e., blood vessels, resident cells, extracellular matrix) can be readily accomplished using a combination of transgenic mice expressing select fluorescent markers, exogenous labeling via fluorescent probes, tissue intrinsic fluorescence, and second/third harmonic generated signals. This protocol describes 1) the preparation of neutrophils for adoptive transfer into the mouse ear, 2) different settings for optimal sub-cellular imaging, 3) strategies to minimize motion artifacts while maintaining a physiological response, 4) examples of membrane remodeling observed in neutrophils using ISMic, and 5) a workflow for the quantitative analysis of membrane remodeling in migrating neutrophils in vivo.

Neutrophil migration relies on the rapid and continuous remodeling of the plasma membrane in response to chemoattractant and its interactions with the extracellular microenvironment. Described herein is a procedure based on Intravital Subcellular Microscopy to investigate the dynamic of membrane remodeling in neutrophils injected in the ear of anesthetized mice.

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first ...

Detecting Migration and Infiltration of Neutrophils in Mice

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