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 be...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 H2O 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 H2O 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&#34; Needle</td>
			<td>BD</td>
			<td>305120</td>
			<td></td>
		</tr>
		<tr>
			<td>26 G x 3/8&#34; 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% H2O2</td>
			<td></td>
			<td></td>
			<td>Mix 1 mL 30% H2O2 with methanol with 9 mL methanol</td>
		</tr>
		<tr>
			<td>3,3&#39;-diaminobenzidine (DAB) kit</td>
			<td>ZSGB-BIO</td>
			<td>ZLI-9018</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G x 1/2&#34; Needle</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>30% H2O2</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 dH2O</td>
		</tr>
		<tr>
			<td>54.8% Percoll</td>
			<td></td>
			<td></td>
			<td>Mix 2.74 mL SIP with 2.26 mL 1&#215;PBS, stand still</td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td></td>
			<td></td>
			<td>Mix 700 mL 100% ethanol with 300 mL dH2O</td>
		</tr>
		<tr>
			<td>70.2% Percoll</td>
			<td></td>
			<td></td>
			<td>Mix 3.51 mL SIP with 1.49 mL 1&#215;PBS, stand still</td>
		</tr>
		<tr>
			<td>80% Ethanol</td>
			<td></td>
			<td></td>
			<td>Mix 800 mL 100% ethanol with 200 mL dH2O</td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td></td>
			<td></td>
			<td>Mix 950 mL 100% ethanol with 50 mL dH2O</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&#39;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&#38;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&#39;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&#215;</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 &#956;L Triton X-100 with 1 L dH2O to get 0.01% Triton X-100</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS), 1&#215;</td>
			<td></td>
			<td></td>
			<td>Mix 90% ddH2O with 10% (v/v) 10&#215;PBS, autoclaved</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS), 10&#215;</td>
			<td></td>
			<td></td>
			<td>Dissolve 16 g NaCl, 0.4 g KCl, 2.88 g Na2HPO4&#183;2H2O, 0.48 g KH2PO4 (anhydrous) in 200 mL ddH2O, 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 dH2O 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 (C6H5O7&#183;H2O) in 200 mL dH2O</td>
		</tr>
		<tr>
			<td>Retrieval Buffer B Stock for IHC</td>
			<td></td>
			<td></td>
			<td>Dissolve 5.88 g trisodium citrate dihydrate (C6H5Na3O7&#183;2H20) in 200 mL dH2O</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&#215;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).
<p class="jove_con...

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

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JoVE Journal

Biology

11.7K Views.  Beijing University of Chinese Medicine. 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.

Video: Detecting Migration and Infiltration of Neutrophils in Mice

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

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 ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

Detecting Establishment of Shared Blood Supply in Parabiotic Mice by Caudal Vein Glucose Injection

Parabiosis is an experimental method for surgically combining two parallel animals along the longitudinal axis of the body. We present a protocol for detecting the successful establishment of blood chimerism in parabionts by a caudal vein injection of glucose. Parabiotic mice were constructed. Glucose was injected into the donor mouse through the tail vein, and the fluctuation of blood glucose level was measured in both mice using a blood glucometer at different time points. Our results showed that after glucose injection, the blood glucose level in donor mice increased sharply after 1 min and decreased slowly thereafter. Meanwhile, the blood glucose level of the recipient mice peaked 15 min after injection. Similar results were obtained with Evans blue, used as a positive control for glucose. The synchronous fluctuation of blood glucose levels indicates that blood flow between the two mice was established successfully.

Here we describe a new method of detecting successful establishment of shared blood circulation of two parabionts through a caudal vein injection of glucose, which causes minimal damage and is not fatal to the parabionts.

Parabiosis is an experimental method for surgically combining two parallel animals along the longitudinal axis of the body. We present a protocol for ...

Isolation of Human Neutrophils from Whole Blood and Buffy Coats

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells recruited at the site of inflammation via rapid extravasation through vessels. There, neutrophils perform an array of functions to kill invading pathogens and mediate immune signaling. Freshly purified neutrophils from human blood are the model of choice for study, as no cell line fully replicates PMN functions and biology. However, neutrophils are short-lived, terminally differentiated cells and are highly susceptible to activation in response to physical (temperature, centrifugation speed) and biological (endotoxin, chemo- and cytokines) stimuli. Therefore, it is crucial to follow a standardized, reliable, and fast method to obtain pure and non-activated cells. This protocol presents an updated protocol combining density gradient centrifugation, red blood cell (RBC) sedimentation, and RBC lysis to obtain high PMN purity and minimize cell activation. Furthermore, methods to assess neutrophil isolation quality, viability, and purity are also discussed.

This protocol details a method for the isolation of neutrophils from whole blood, buffy coats, or leukapheresis membranes, achieving good yield, high purity, and minimal cell activation. We utilize gradient purification, red blood cell (RBC) sedimentation, and RBC lysis to obtain a high-quality/purity neutrophil preparation.

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells ...