This work was supported by NIH grant P20 GM109098 and the Innovation Award Program from Praespero to Edwin Wan. Flow Cytometry experiments were performed in the WVU Flow Cytometry &#38; Single Cell Core Facility, which is&#160;supported by NIH grants S10 OD016165, U57 GM104942, P30 GM103488, and P20 GM103434.

All protocols and procedures performed were approved by the Institutional Animal Care and Use Committee (IACUC) of West Virginia University. The mice were housed under specific-pathogen-free conditions in the vivarium at West Virginia University. &#160;
1. Preparation of solutions
<ol>
	<li>Prepare perfusion buffer (phosphate buffered saline, PBS). Use approximately two 10 mL aliquots of ice-cold PBS per mouse.</li>
	<li>Prepare lung cell medium/FACS buffer. FACS buffer contains PBS supplemented with 1% fetal bovine serum (FBS). Keep the medium cold for the entire process of lung excision and transfer. Prepare approximately 8 mL per lung sample. Prepare fresh medium/FACS buffer prior to the experiments.</li>
	<li>Prepare dissociation buffer for single cell isolation. Buffer contains 1 mg/mL collagenase D and 200 &#181;g/mL DNase I in Hank&#8217;s buffered salt solution (HBSS). Prepare fresh from the stock solution (100 mg/mL collagenase D and 10 mg/mL DNase I) prior to the experiments. Approximately 6 mL per lung sample is needed. Ensure buffer reaches room temperature prior to the use.</li>
	<li>Prepare homogenization buffer for lung tissue homogenization. Buffer contains PBS and 1x proteinase and phosphatase inhibitor cocktail (stock = 100x). Freshly prepare prior to the experiments. Keep the buffer cold during the entire homogenization process. Approximately 200 &#181;L of the buffer is sufficient for homogenizing a single right lobe of the lungs.</li>
</ol>
2. Transient middle cerebral artery occlusion (tMCAO)
NOTE: Procedures for tMCAO via monofilament insertion to the middle cerebral artery were documented in detail previously10. In these experiments, 8- to 12-week-old male C57BL/6J mice, weighing 25-30 g, were used.
<ol>
	<li>In brief, subcutaneously administer 2 mg/kg meloxicam to the mice before surgery. Deeply anesthetize the mice in an induction chamber using 5% isoflurane. Confirm deep anesthetization using the toe-pinch method. Maintain anesthesia with 1-2% isoflurane during the surgery using a nose cone. Maintain the body temperature at 36.5 - 38 &#176;C throughout the procedure by placing a warm blanket underneath the mice. All surgical tools should be autoclave-sterilized a day prior to the surgery.</li>
	<li>Shave and prepare the skin on the ventral neck using 70% ethanol followed by chlorhexidine scrub. Subcutaneously administer 2 mg/kg bupivacaine over the incision area prior to making the first incision. Use eye ointment to cover the eyes to prevent dryness. Make a midline neck incision. Gently pull aside soft tissues around the trachea.</li>
	<li>Identify the left common carotid artery and the external carotid artery.</li>
	<li>Apply a temporary suture (size 6/0) to the common carotid artery to stem the flow of the blood. Make an incision in the external carotid artery.</li>
	<li>Insert a silicone rubber-coated monofilament (size 6-0) into the external carotid artery, then advance the monofilament to the middle cerebral artery. Leave the monofilament in the middle cerebral artery for 60 min (occlusion).</li>
	<li>Monitor the rate of blood flow using Laser Doppler Flowmetry. A reduction in the flow rate to the middle cerebral artery that is &#62; 80% indicates a successful occlusion.</li>
	<li>Following the 60 min of occlusion, remove the monofilament to allow for the reperfusion to occur. Close the incision.</li>
	<li>In sham-operated mice, perform steps 2.1-2.6 without the insertion of the monofilament. In these mice, the rate of blood flow should remain steady during the entire procedure.</li>
	<li>Monitor the mice daily and measure the neurological deficits using standard scoring criteria11. 0 &#8211; no neurological deficit; 1 &#8211; retracts contralateral forepaw when lifted by the tail; 2- circles to the contralateral when lifted by the tail; 3- falling to the contralateral while walking; 4 &#8211; does not walk spontaneously or is comatose; 5 &#8211; dead.</li>
	<li>Provide subcutaneous injections of normal saline and analgesic (meloxicam, 2 mg/kg) every 24 hours for 3 days following the surgery.</li>
	<li>Isolate the lungs 24 and 72 h following tMCAO for the downstream analysis.</li>
</ol>
3. Harvesting the lung tissues
<ol>
	<li>Deeply anesthetize the mouse by intraperitoneal (i.p.) injection of 100 mg/kg of ketamine and 10 mg/kg of xylazine. Confirm deep anesthetization using the toe-pinch method.</li>
	<li>To perform the whole body transcardial perfusion12, pin the mouse to the surgical platform and wet fur with 70% ethanol to reduce fur distribution into the body cavity. Use fine dissection scissors to make a transverse incision in the caudal abdomen and then a midline incision of the peritoneum, stopping at the thoracic cavity prior to piercing the diaphragm.
	<ol>
		<li>Do not cut through the muscle/hide into the peritoneal cavity and do not continue incision past abdomen.</li>
	</ol>
	</li>
	<li>Stop the incision as soon as the thoracic cavity has been reached before puncturing the diaphragm. Take care not to damage the heart and lungs which may compromise perfusion quality or cell recovery</li>
	<li>Using fine dissection scissors make a midline incision of the peritoneum stopping at the thoracic cavity prior to piercing the diaphragm.</li>
	<li>Grasp the mouse by the distal end of the sternum using forceps and lift upward while cutting through the diaphragm being certain not to damage the heart, lungs, or vasculature. Once organs are visible make an incision through the rib cage and separate and pin back the rib case so that the heart and lungs are fully exposed.</li>
	<li>Carefully make a small incision in the right atrium near the aortic arch. This will serve as the outflow for the perfusion.</li>
	<li>Immediately after, gently insert a 25 G x 5/8 needle attached to a syringe filled with 10 mL of cold PBS into the distal superior surface of the left ventricle. Use care not to insert through the ventricular septum into the right ventricle. Needle should be barely inserted into the left ventricle. 
	NOTE: If ventricular septum is pierced, fluid will rush out from the mouse&#8217;s nose. This will decrease the perfusion quality and result in cell loss.</li>
	<li>If desired, a clamp may be used to hold the needle securely in place in the left ventricle during the perfusion.</li>
	<li>Steadily but gently push 10 mL of cold PBS into the heart. Mouse tissue should begin to perfuse and clear off the blood as fluid exits from the right atrium.
	<ol>
		<li>Push a second aliquot of 10 mL of PBS until sufficient blood clearance has occurred. Liver will have a light brown appearance and lungs will transition from a reddish pink to mostly white in color.</li>
	</ol>
	</li>
	<li>Using forceps and fine dissection scissors, carefully remove all surrounding tissues including the heart, trachea, esophagus, thymus, connective tissue, lymph nodes, and large bronchials.</li>
	<li>Separate individual lung lobes and place the lobes into a Petri dish on ice containing a 1-2 mL aliquot of cold lung cell medium.</li>
</ol>
4. Homogenization of lung tissue for multiplex bead arrays using bead homogenizer
<ol>
	<li>Pre-chill a 2 mL conical screw cap tube containing 3 sterile 2.3 mm zirconia/silica beads. Add 200 &#181;L of cold homogenization buffer to the tube.</li>
	<li>Weigh the lobe, transfer the lobe to the pre-chilled conical tube containing the beads and homogenization buffer. 
	NOTE: Approximately 50-100 mg of tissue homogenized in 200 &#181;L of buffer will be sufficient for detecting cytokine and chemokine expression in the sample.
	<ol>
		<li>Homogenize the lobe using a bead-based homogenizer (see Table of Materials) at 4,000 rpm for 2 min.</li>
		<li>Ensure the tissue is completely homogenized. Increase the time if needed.</li>
	</ol>
	</li>
	<li>Following homogenization, centrifuge the tube in a pre-chilled (4 &#176;C) micro-centrifuge at 15,870 x g for 3 min.</li>
	<li>Transfer the supernatants to a pre-chilled 1.5 mL microtube.</li>
	<li>Place the sample directly on ice for immediate use or store the sample at -80 &#176;C for future use. Avoid multiple freeze-thaw cycles.</li>
</ol>
5. Lung tissue dissociation and single cell isolation
<ol>
	<li>Pour the lung cell medium off, of the remaining lung lobes to avoid dilution of the dissociation buffer. Then, carefully insert a 1 mL syringe with 25G and 5/8 needle containing 1 mL of the dissociation buffer into lung tissue. &#160;</li>
	<li>Inject small fractions (approximately 1/4th to 1/5th) of the dissociation buffer into each lung lobe and gently inflate. 
	NOTE: Most of the buffer will rush out soon after inflating as the lobe has been excised.</li>
	<li>Repeat the injection of dissociation buffer 1-2x.</li>
	<li>Incubate the lung lobes with dissociation buffer for 2 min at room temperature.</li>
	<li>Finely mince the lung lobes into small pieces using fine dissection scissors.</li>
	<li>Transfer the minced lung pieces with the dissociation buffer to a 15 mL centrifuge tube. &#160;Supplement with additional dissociation buffer to a total of 6 mL. Vortex vigorously for 1 min.</li>
	<li>Incubate the sample for 45 min at 37 &#176;C. Vortex vigorously every 7-8 min.</li>
	<li>After 45 min of incubation, vortex the sample vigorously for 1 min for optimal results.</li>
	<li>Further dissociate the sample by passing through a 100 &#181;m cell strainer to remove residual, undesired connective, and interstitial tissue.</li>
	<li>Centrifuge the sample for 10 min at 380 x g, discard the supernatant.</li>
	<li>Add 5 mL of cold lung cell medium to the sample. Resuspend the cell pellet by gentle vortexing.</li>
	<li>Centrifuge the sample for 10 min at 380 x g, discard the supernatant.</li>
	<li>Add 1 mL of cold lung cell medium. Resuspend the cell pellet by gentle vortexing.</li>
	<li>Pass through a 100 &#181;m cell strainer, and count cells.</li>
</ol>
6. Flow cytometric analysis for the lung immune cell niche
<ol>
	<li>Seed 1-2 x 106 cells/antibody set into a 96 well round bottom plate (3 sets in total). Centrifuge the cells for 3 min at 830 x g. Discard supernatants.</li>
	<li>Resuspend the cell pellet in 50 &#181;L of cold PBS containing fixable live/dead stain. Prepare the stain according to the manufacturer&#8217;s instructions.</li>
	<li>Incubate the cells at 4 &#176;C for 20 min protected from light. Centrifuge the cells for 3 min at 830 x g. Discard supernatants.&#160;&#160;</li>
	<li>Resuspend the cell pellet with 25 &#181;L of cold FACS buffer containing 5 &#181;g/mL of anti-mouse CD16/32 antibody. Incubate at 4 &#176;C for 10-15 min.</li>
	<li>Add 25 &#181;L of cold FACS buffer containing antibody combinations listed in Table 1 to the cells. Mix by gentle pipetting. 
	NOTE: Total volume of FACS buffer for the antibody staining is 50 &#181;L. Therefore, when preparing a master mix of antibodies in 25 &#181;L, the concentration of antibodies should be double the final concentration. &#160;</li>
	<li>Incubate the cells at 4 &#176;C for 20 min protected from light. Centrifuge the cells for 3 min at 830 x g. Discard supernatants.</li>
	<li>Resuspend the cells with 100 &#181;L of cold FACS buffer. Centrifuge the cells for 3 min at 830 x g. Discard supernatants.</li>
	<li>Resuspend the cells with 100 &#181;L of cold FACS buffer. Transfer to a polystyrene round-bottom 5 mL FACS tube containing 150 &#181;L of additional FACs buffer. Analyze the sample immediately using a flow cytometer. 
	NOTE: Cell fixation using the following steps allows samples to be analyzed later.</li>
	<li>Following step 6.7., resuspend the sample with 100 &#181;L of 1% paraformaldehyde in PBS.</li>
	<li>Incubate the sample at 4 &#176;C for 15 min protected from light. Centrifuge the cells for 3 min at 830 x g. Discard supernatants.</li>
	<li>Resuspend the cells with 100 &#181;L of cold FACS buffer. Centrifuge the cells for 3 min at 830 x g. Discard supernatants.</li>
	<li>Resuspend the cells with 100 &#181;L of cold FACS buffer. Store the cells at 4 &#176;C, protected from light. Analyze the sample within 72 h.</li>
</ol>
7. Multiplex bead arrays for cytokine and chemokine detection
NOTE: Commercially available proinflammatory chemokine and inflammation multiplex panels (see Table of Materials) were used to determine the expression of chemokines and cytokines in the lungs following tMCAO induction according to manufacturer protocol, which has been described in detail13.
<ol>
	<li>In brief, thaw the analytes on ice if they were stored at -80 &#176;C after tissue homogenization. Centrifuge the sample at 590 x g for 2 min at 4 &#176;C before use to remove any residual tissue.</li>
	<li>Generate a standard curve by serially diluting the standard cocktail (C7) which is at a known concentration of 10 ng/mL into 7 standards (C1-6) with the last standard being assay buffer alone (C0).</li>
	<li>Once all reagents have warmed to room temperature, add 25 &#181;L of each standard and 25 &#181;L of assay buffer to a V bottom 96 well plate.&#160;</li>
	<li>To each sample well, add 25 &#181;L of lung homogenate (analyte) and 25 &#181;L of assay buffer to a V bottom 96 well plate</li>
	<li>Vortex the pre-coated beads vigorously, then add 25 &#181;L of pre-coated beads to each of the standard and sample wells.</li>
	<li>Seal the plate and protect from light by covering with foil. Shake the plate at 110 rpm for 2 h at room temperature.</li>
	<li>Centrifuge at 230 x g for 5 min. Add 200 &#181;L of wash buffer at 1x (stock solution is 20x, dilute to 1x with water). Incubate for 1 min.</li>
	<li>Centrifuge at 230 x g for 5 min. Resuspend the standard and sample with 25 &#181;L detection antibodies.</li>
	<li>Seal and cover the plate to protect from light. Shake plate at 110 rpm for 1 h at room temperature.</li>
	<li>Add 25 &#181;L of streptavidin-phycoerythrin (SA-PE) to each standard and sample well.</li>
	<li>Seal and cover the plate to protect from light. Shake at 110 rpm for 30 min at room temperature.</li>
	<li>Spin the plate at 230 x g for 5 min. Resuspend the standard and samples in 1x wash buffer.</li>
	<li>Transfer to a well-labeled polystyrene round-bottom 5 mL FACS tube containing an additional 150 &#181;L of 1x wash buffer.</li>
	<li>Determine the PE signal fluorescence intensity for the standards and samples using a flow cytometer.</li>
	<li>Construct a standard curve for each chemokine and cytokine using the mean fluorescence intensity (MFI) of PE against the pre-determined concentrations.</li>
	<li>Determine the concentration of each chemokine and cytokine using the standard curve and the MFI from each sample. The multiplex panel provides data analysis software that can be used to determine the concentration of each analyte. The weight of the apical/superior lobe is used to determine the concentration of cytokines or chemokine per milligram of tissue. &#160;&#160;</li>
</ol>

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The authors have nothing to disclose.

The protocols described here allow for the identification of lung immune cell types and the expression of chemokines or cytokines in the same mouse. If a histopathology study is desired, an individual lobe can be removed and fixed for that purpose prior to proceeding to the single cell isolation steps. One limitation of this method is that this approach may not be suitable in some disease settings if the change in the immune cell composition and the expression of chemokines and/or cytokines are anticipated to be unequall...

The lungs are a barrier organ, exposed to the external environment and, therefore, are constantly receiving immunological challenges such as pathogens and allergens1. The activation of lung-resident immune cells and the infiltration of immune cells from the periphery are required to clear pathogens from the pulmonary environment. Additionally, lung-resident immune cells maintain tolerance to commensal bacteria, suggesting that these cells play a role in pathogen clearance and maintaining homeostasis1. Alveolar and interstitial macrophages are among the lung-resident sentinel immune cells that sense pathogens via pattern recognition receptors and clear these pathogens by phagocytosis2. Lung-resident dendritic cells bridge the innate and adaptive immune response through antigen presentation3. In addition, activated local innate immune cells produce cytokines and chemokines that amplify the inflammatory response and stimulate the infiltration of immune cells such as monocytes, neutrophils, and lymphocytes into the lungs1. Ischemic stroke has been shown to modify systemic immunity and lead to increased susceptibility to pulmonary infection; however, few studies have evaluated the pulmonary compartment following ischemic stroke, though some studies have examined it during &#160;inflammatory conditions4,5,6,7,8,9. The goal of the methods described herein is to simultaneously determine lung pathology, immune cell composition, and the levels of cytokine and chemokine expression in the lungs to evaluate alterations to the pulmonary compartment and assess potential alterations to the pulmonary immune response following ischemic attack.
Described here is a protocol for obtaining single cell suspensions from the lungs of the mice to identify 13 types of immune cells. This protocol is based on tissue digestion with collagenase D without the need of an automated tissue dissociator. Additionally, we developed a protocol to prepare tissue homogenates that can be used to determine the expression levels of 13 different cytokines or chemokines using flow cytometry-based multiplex bead arrays. This protocol was successfully used to investigate the effects of ischemic stroke on pulmonary immunity and can be used in other disease models as well.

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			<td height="21" style="height:21px;width:289px;">B220-APC, clone RA3-6B2</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">103212</td>
			<td style="width:412px;">1:200 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Beadbug 3 position bead homogenizer</td>
			<td style="width:129px;">Benchmark Scientific</td>
			<td style="width:101px;">D1030</td>
			<td>Tissue homogenizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CCR2-BV421, clone SA203G11</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">150605</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD103-BV421, clone 2E7</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">121422</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD11b-PE/Cy7, clone M1/70</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">101216</td>
			<td>1:400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD11c-PE/Cy7, clone N418</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">117318</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD11c-Percp/Cy5.5, clone N418</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">117328</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD4-BV421, clone GK1.5</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">100443</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD45-FITC, clone 30-F11</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">103108</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD64-APC, clone X54-5/7.1</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">139306</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD8-PE, clone 53-6.7</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">100708</td>
			<td>1:800 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Collagenase D</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td>11088882001</td>
			<td>Component in the dissociation buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Conical screw cap tube</td>
			<td style="width:129px;">ThermoFisher</td>
			<td style="width:101px;">02-681-344</td>
			<td>Tube for tissue homogenization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">DNase I</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td>10104159001</td>
			<td>Component in the dissociation buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Fc block CD16/32 antibody</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">101320</td>
			<td>1:100 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">genlteMACS dissociator</td>
			<td style="width:129px;">Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>Comparsion of lung digestion with or without mechanical dissociator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">gentleMACS C tubes</td>
			<td style="width:129px;">Miltenyi Biotec</td>
			<td style="width:101px;">130-093-237</td>
			<td>Tube for tissue disscoiation with genlteMACS dissociator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Halt protease and phosphatase inhibitor cocktial</td>
			<td style="width:129px;">ThermoFisher</td>
			<td style="width:101px;">78442</td>
			<td>Component in the homogenization buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Laser doppler monitor</td>
			<td style="width:129px;">Moor</td>
			<td style="width:101px;">MOORVMS-LDF</td>
			<td>Blood flow monitoring during tMCAO</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">LEGENDplex proinflammatory chemokine panel</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">740451</td>
			<td>Multiplex bead array</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">LIVE/DEAD fixable near-IR stain</td>
			<td style="width:129px;">ThermoFisher</td>
			<td style="width:101px;">L34976</td>
			<td>Use for dead cell exclusion during flow cytometric analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Ly6C-PE, clone HK1.4</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">128008</td>
			<td>1:800 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Ly6G-BV510, clone 1A8</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">127633</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">MCAO suture L56 reusable 6-0 medium</td>
			<td style="width:129px;">Doccol</td>
			<td style="width:101px;">602356PK10Re</td>
			<td>tMCAO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">MHC II-BV510, clone M5/114.15.2</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">107636</td>
			<td>1:800 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">NK1.1-Percp/Cy5.5, clone PK136</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">108728</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Siglec F-PE, clone E50-2440</td>
			<td style="width:129px;">BD Biosciences</td>
			<td style="width:101px;">552126</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Silk suture thread, size 6/0</td>
			<td style="width:129px;">Fine Science Tools</td>
			<td style="width:101px;">18020-60</td>
			<td>tMCAO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">SomnoSuite anesthesia system</td>
			<td style="width:129px;">Kent Scientific</td>
			<td style="width:101px;">SS-01</td>
			<td>Mouse anaesthetization for tMCAO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">TCRb-BV510, clone H57-897</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">109234</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Zirconia/silica beads, 2.3 mm</td>
			<td style="width:129px;">Biospec</td>
			<td style="width:101px;">11079125z</td>
			<td>Beads for tissue homogenization</td>
		</tr>
	</tbody>
</table>

characterization of immune cells and proinflammatory mediators in the pulmonary environment

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be altered by severe brain injury. This is evidenced by the fact that pneumonia is a major cause of mortality in patients who have suffered from ischemic stroke. The goal of this protocol is to describe the use of multicolor flow cytometric analysis to identify 13 types of immune cells in the lungs of mice, including alveolar macrophages, interstitial macrophages, CD103+ or CD11b+ dendritic cells (DCs), plasmacytoid DCs, eosinophils, monocytes/monocyte-derived cells, neutrophils, lymphoid-derived&#160; T and B cells, NK cells, and NKT cells, following ischemic stroke induction by transient middle cerebral artery occlusion. Moreover, we describe the preparation of lung homogenates using a bead homogenization method, to determine the expression levels of 13 different cytokines or chemokines simultaneously by multiplex bead arrays coupled with flow cytometric analysis. This protocol can also be used to investigate the pulmonary immune response in other disease settings, such as infectious lung disease or allergic disease.

We recently reported that ischemic stroke induction in mice alters the immune cell composition of the lungs11. Specifically, transient cerebral ischemia increased percentages of alveolar macrophages, neutrophils, and CD11b+ DCs, while diminishing percentages of CD4+ T cells, CD8+ T cells, B cells, NK cells, and eosinophils in the pulmonary compartment. Moreover, cellular alteration corresponded to significantly diminished levels of multiple chemokines in the lungs. Described here is a method for t...

Watch this Scientific Journal Video about Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment at JoVE.com

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

This protocol describes the use of flow cytometry to identify the changes in immune cell composition, cytokine profile, and chemokine profile in the pulmonary environment following transient middle cerebral artery occlusion, a murine model of ischemic stroke.

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be ...

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Research

JoVE Journal

Immunology and Infection

3.2K Views.  West Virginia University. This protocol describes the use of flow cytometry to identify the changes in immune cell composition, cytokine profile, and chemokine profile in the pulmonary environment following transient middle cerebral artery occlusion, a murine model of ischemic stroke.

Video: Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...