Name: Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles (Video) | JoVE Journal
Uploaded: 2012-06-08T12:00:00.000+00:00
Duration: 11 min 26 s
Description: Learn to harvest murine alveolar macrophages via bronchoalveolar lavage and evaluate their activation by polyanhydride nanoparticles using flow cytometry. This protocol enables immunology and nanotechnology research.

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5 minutes

The authors would like to thank the U.S. Army Medical Research and Materiel Command (Grant Numbers W81XWH-09-1-0386 and W81XWH-10-1-0806) for financial support and Dr. Shawn Rigby from the Iowa State University Flow Cytometry Facility for his expert technical assistance.

1. Harvesting Alveolar Macrophages (AMɸ) from the Mouse using Lung Lavage
<ol>
 <li>Wear appropriate personal protective equipment (PPE) such as a lab coat, disposable gloves, and proper eye protection.</li>
 <li>Prepare complete AMɸ (cAMɸ) medium before initiation of harvest. Add 2.5 mL of penicillin/streptomycin (pen/strep) solution, 250 μL of β-mercaptoethanol (2-mercaptoethanol), and 25 mL of heat inactivated fetal bovine serum (FBS) to 222.25 mL of Dulbelcco's Modified Eagle Medium (DMEM). Filter-sterilize cAMɸ medium using a 0.2 μm pore size bottle top filter under vacuum.</li>
 <li>Euthanize a healthy mouse by CO2 asphyxiation in an euthanization chamber. Although a C57BL/6 mouse (6-8 weeks old) was used for the purposes of this project, this lung lavage technique can be performed on any strain of mouse. Using a compressed gas cylinder, let gas flow into the chamber for 1 min prior to placing the mouse in the chamber to assure that the chamber is fully charged with CO2. Place the mouse in the chamber for at least one minute to euthanize the mouse. In addition, exsanguinate each mouse via cardiac puncture as a physical method to confirm mouse death as described in 1.4.</li>
 <li>Lay mouse on its back (dorsal side), spray with 70% ethanol, locate the sternum and, at a 30 degree angle from the abdomen, insert a 23 gauge needle (attached to a 1 mL syringe) about 50 mm into the thoracic cavity. With proper placement of the needle, it should be possible to collect between 0.4 to 1 mL of blood depending on the size of the mouse. Examine the animal for cessation of vital signs.</li>
 <li>Place mouse on lab bench so that it is lying on its abdomen (ventral side). Spray mouse with 70% ethanol to sterilize the working environment. Pinch skin about halfway down the spine of the mouse. Using dissection scissors that have been sterilized with 70% ethanol, make a small incision through the skin.</li>
 <li>Grab the skin on both sides of the incision and pull the skin in opposite directions along the cranial-caudal axis of the mouse. When done correctly, the skin will be removed from the underlying carcass.</li>
 <li>The neck of the mouse should now show exposed muscles and glands. Carefully cut that tissue and pull it towards the anterior of the mouse using forceps, thereby exposing the larynx, trachea, and esophagus of the mouse. Avoid cutting the jugular vein or carotid artery.</li>
 <li>The trachea is now visible and is identifiable as the tissue with cartilaginous rings around it. Delicately hold the trachea with forceps and gently lift and separate the trachea from the esophagus, which is located on the dorsal side.</li>
 <li>Make a small incision anterior to where one holds the trachea but posterior to the larynx. The incision should be made at approximately a 45° degree angle. Also, do not completely transect the trachea, as this will prevent it from retracting into the body cavity.</li>
 <li>Fill a 1 cc syringe fitted with a Tom Cat catheter with sterile PBS in preparation to insert the catheter into the lumen of the trachea. Hold the trachea using forceps and with your hand hold the catheter close to the tip to increase control and gently insert it into the tracheal incision site. Gently insert catheter into trachea about 20 mm. Grip the trachea that is around the catheter tube, freeing the other hand.</li>
 <li>Using a 1 mL syringe fitted with the catheter, gently infuse 0.75 mL of room temperature, sterile PBS into the lungs and then gently aspirate the PBS back into syringe. Repeat this process three times while externally massaging the chest. Disconnect the syringe from the catheter and dispense the lavage fluid into a 15 mL centrifuge tube. After filling the syringe with 0.75 mL of fresh PBS and reconnecting the syringe to the catheter, repeat steps 1.9 and 1.10 twice for each mouse. Fluid recovery may be partial, obtaining less than the volume infused. If collecting cells from more than one mouse, place the 15 mL tube with the harvested cells on ice until all lung lavages have been performed.</li>
</ol>
2. Processing of Lung Lavage AMɸ Harvest
<ol>
 <li>Centrifuge lung lavage fluid at 250 x g for 10 min at 4 °C.</li>
 <li>Using sterile technique, decant supernatant into appropriate waste container inside a biosafety cabinet. Resuspend the cell pellet by gently raking the centrifuge tube across the top of a test tube rack. Add 1 mL of cAMɸ medium to resuspend the cells.</li>
 <li>Enumerate the cells using a Coulter Particle Counter Z1 by pipetting 20 μL of cells into 10 mL of Isoton II Diluent in Coulter counter vial. Add 3 to 4 drops of Zap-oglobin II lytic reagent into vial, gently invert 3 to 4 times and then load the sample vial onto the counter to obtain a cell concentration. Be sure to program the counter with a dilution factor of 2 x 104 to display the cell concentration as the number of cells per mL.</li>
 <li>Dilute cells to 2.5 x 105 per mL using cAMɸ medium.</li>
 <li>Dispense 2 mL of 2.5 x 105 cells/mL into each well of a 6-well tissue culture dish.</li>
 <li>To enrich for AMɸ, incubate culture dishes inside a 37 °C humidified incubator with a 5% CO2 atmosphere for 6 h to allow cells to adhere to the bottom of the well. Gently aspirate the supernatant as to not disrupt adherent cells.</li>
 <li>Rinse the wells using sterile PBS (calcium and magnesium free) to remove non-adherent cells and debris. Add 2 mL of cAMɸ medium to each well.</li>
 <li>Incubate cells overnight inside a 37 °C humidified incubator with a 5% CO2 atmosphere prior to addition of stimulants. After this enrichment step, approximately 87% of the cells are positive for the macrophage markers CD11b and F4/80 (Figure 2c).</li>
</ol>
3. Addition of Polyanhydride Nanoparticles and Control Treatments
<ol>
 <li>Fabricate nanoparticles via polyanhydride anti-solvent nanoencapsulation15. In this process, the polymer is dissolved in methylene chloride (4 °C at a concentration of 25 mg/mL) and precipitated in pentane (-20 °C at a ratio 1:200 methylene chloride:pentane).</li>
 <li>Weigh out polyanhydride nanoparticles using sterilized weigh paper on a balance that can accurately measure microgram amounts. Add particles to a 1.5 mL microcentrifuge tube.</li>
 <li>Resuspend the nanoparticles in ice cold cAMɸ. Calculations are made such that no more than 0.3 mL of medium is added to each well to obtain the desired concentration of nanoparticles. Keep the resuspended particles on ice until the nanoparticles are added to the culture well.</li>
 <li>Prior to adding the nanoparticles to the AMɸ cultures, sonicate particles using a bench top sonicator with a microtip. Sterilize microtip by spraying with 70% ethanol and wiping with a sterile Kimwipe prior to sonication. Sonicate at 10-20 joules for 30 to 45 s while keeping the tube on ice. Observe nanoparticle suspension macroscopically. If particles are not sufficiently dispersed, allow the suspension to set on ice for 1 to 2 min to ensure that the nanoparticles are below the glass transition temperature of the polymer before sonicating again for 30 to 45 sec. The intent of this sonication step is to adequately disperse the nanoparticles, as the 50:50 CPTEG:CPH nanoparticles tend to aggregate when exposed to an aqueous environment. However, some small aggregates of nanoparticles may still remain in the suspension.</li>
 <li>Nanoparticle suspension (adequately dispersed) should be kept on ice and be used immediately to prevent premature surface erosion or aggregation.</li>
 <li>Remove the culture dishes containing the AMɸ from incubator and place in the biosafety cabinet. Tilt plate at approximately a 45 degree angle and pipette off the amount of medium that will be added to the nanoparticle suspension (it should not exceed 0.3 mL). Do not disturb the adherent cells when removing the medium.</li>
 <li>Vortex the polyanhydride nanoparticle suspension briefly prior to adding it to the appropriate wells. Pipette the amount of nanoparticle suspension (no more than 0.3 mL) into the specific well(s); however, do not pipette up and down to mix. This action could detach the adherent AMɸ from the well. The particles will settle to the bottom of the well and contact the cells. For the studies outlined in this report, the final concentration of nanoparticles co-cultured with the AMɸ was 125 μg/mL. Proceed with subsequent wells and desired treatment groups. Include positive and negative control groups, such as medium only and 200 ng/mL lipopolysaccharide (LPS) to designated wells.</li>
 <li>Return the culture dishes to the 37 °C humidified incubator with a 5% CO2 atmosphere for 48 h. Kinetic studies in our laboratory have demonstrated 48 hours to be the optimal culture period for evaluating both cell-surface marker expression and cytokine secretion as it permits adequate time for transcription and protein synthesis to occur. Although greater levels of cytokines accumulate during an extended culture period (i.e., 72 hr), evaluating cell surface marker expression at this time point can be confounded by the contribution of the elevated levels of cytokines. The changes in surface marker expression observed may not be directly attributable to stimulation with the nanoparticles themselves.</li>
</ol>
4. Evaluating AMɸ Activation using Flow Cytometry
<ol>
 <li>Prepare fluorescence-activated cell sorting (FACS) buffer (0.1 % sodium azide and 0.1 % bovine serum albumin (BSA)) in 0.1 M phosphate buffered saline (pH 7.4) prior to harvesting the plated macrophages that have been co-cultured with nanoparticles or stimulants.</li>
 <li>Prepare working solutions of the blocking buffer and the monoclonal antibodies. To prepare the block buffer, dilute rat IgG (Sigma) and anti-CD16/32 (eBioscience) to 1 mg/mL and 10 μg/mL, respectively, in FACS buffer. Dilute surface marker antibody panel in FACS buffer to either the manufacturer recommended concentration or the user optimized concentration. For the results presented in this work, the surface marker panel consists of antibodies against MHC II, CD86, CD40 and CIRE (CD209). Anti-mouse antibodies against the macrophage markers F4/80 and CD11b are also included in the panel to positively identify the macrophage phenotype during data analysis. Care must be taken when selecting antibodies against MHC II molecules, as inbred mouse strains often differ in their MHC II haplotypes. The antibodies selected must react with the specific MHC alloantigens expressed by your mouse strain of interest.</li>
 <li>After 48 h of incubation with nanoparticles and/or stimulants, place cell culture dishes on ice for 20 min. To remove the adherent macrophages from the plates, gently scrape wells with a 24 cm cell scraper.</li>
 <li>Using a 5 mL serological pipette, gently aspirate the cells and medium up and down five times to generate a single cell suspension. Pipette the contents of individual wells into separate 5 mL polystyrene tubes so as not to mix together cells from different experimental treatments.</li>
 <li>Add 2 mL of FACS buffer to each tube.</li>
 <li>Centrifuge tubes of cells at 250 x g at 4 °C for 10 min.</li>
 <li>Decant supernatant and gently resuspend cell pellet in residual fluid by raking the tubes across the top of a test tube rack.</li>
 <li>In order to prevent non-specific binding of the monoclonal antibodies, add 10 μL of blocking buffer (prepared in 4.2) to each tube of cells. Vortex tubes and incubate on ice for 30 min.</li>
 <li>Add appropriate volume of diluted monoclonal antibodies to each tube and incubate in the dark on ice for 15 min. Tubes that contain samples to be analyzed should receive the combination of antibodies of the polycromatic panel (previous optimization of combination of antibodies should be performed by individual labs). Additional control groups should be included to correctly set the acquisition parameters on the flow cytometer, including a tube of cells that were not labeled, and compensation tubes that correspond to labeled cells using each single color separately. To correctly set the gates for flow cytometric analysis, the Fluorescence Minus One (FMO) controls should be included during acquisition. FMO controls are cells labeled with all the fluorochrome-conjugated antibodies that are used on the panel except one that is replaced by its respective isotype control. Positive and negative populations (i.e., separate aliquots of cells treated with a positive control stimulant and medium alone) should also be included in FMO and compensation control tubes.</li>
 <li>Add 2 mL of FACS buffer to each tube, vortex and centrifuge at 250 x g at 4 °C for 10 min.</li>
 <li>Decant supernatant and resuspend cell pellet in residual fluid by raking the tubes across the top of a test tube rack.</li>
 <li>If the antibody panel consists of an antibody not directly conjugated to a fluorochrome but is instead conjugated to biotin, a fluorochrome-conjugated streptavidin is needed to visualize the surface marker. In this step, add an appropriate volume of the diluted fluorochrome-conjugated streptavidin and incubate in the dark for 15 min on ice. As in 4.2, the streptavidin will be diluted to a concentration recommended by the manufacturer or to one optimized by the user. This step is only needed for panels containing biotinylated primary antibodies.</li>
 <li>Perform a wash step as described in 4.10.</li>
 <li>Dilute an appropriate volume of BD stabilizing fixative 1:3 in deionized water. Add 100 μL of diluted fixative to each tube while gently vortexing to prevent clumping of cells. Labeled and fixed cells can be stably stored in the dark at 4 °C for approximately one week prior to acquisition on a flow cytometer.</li>
</ol>
5. Representative Data
Nanoparticles fabricated in step 3.1 had an average diameter of 163 ± 24 nm and a morphology consistent with that obtained in previous studies5,16. Nanoparticles in solution prior to and after sonication are shown in Figure 1 to demonstrate the need for sonication to ensure adequate dispersal of the particles. Flow cytometric analysis of harvested AMɸs obtained via lung lavage is shown in Figure 2. Labeling cells with a combination of anti-mouse CD11b and anti-mouse F4/80 antibodies as well as the corresponding FMO controls allows for establishing background labeling, identifying AMɸs and gating for further analysis. Treatment of alveolar macrophages with polyanhydride nanoparticles enhances activation as shown by the increased mean fluorescence intensity of MHC II, CD40, CD86, and CIRE (Figure 3).
<img alt="Pink solution in microcentrifuge tubes; protein precipitation experiment result comparison." src="/files/ftp_upload/3883/3883fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. 50:50 CPTEG:CPH nanoparticles prior to (A) and after (B) 30 s of sonication.
<img alt="Flow cytometry diagram showing CD11b and F4/80 isotype analysis; data points indicate cell markers." src="/files/ftp_upload/3883/3883fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Flow cytometric analysis of harvested alveolar macrophages labeled with (A) Alexa Fluor 700 anti-CD11b and the PE-Cy7 FMO Control, (B) Alexa Fluor 700 FMO Control and the PE-Cy7 anti-F4/80 and (C) Alexa Fluor 700 anti-CD11b and PE-Cy7 anti-F4/80. The number in the top right corner represents the percentage of double-positive cells.
<img alt="Flow cytometry histogram charts of MHC II, CD40, CD86, CIRE; immune response analysis." src="/files/ftp_upload/3883/3883fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. Histograms demonstrating an increase in the fluorescence intensity for surface expression of MHC II (A), CD40 (B), CD86 (C) and CIRE (D) after co-culture of alveolar macrophages with 50:50 CPTEG:CPH polyanhydride nanoparticles for 48 hr. Histograms depict results for AMɸ labeled as FMO controls (<img alt="Atomic structure, electron configuration, Bohr model diagram, educational physics concept." src="/files/ftp_upload/3883/3883line1.jpg" data-altupdatedat="1787630408000" data-alt="Graph Line 1">), untreated AMɸ labeled with antibodies against all cell surface markers (<img alt="Chromatography; color separation, four green bands; chemical analysis, lab experiment diagram." src="/files/ftp_upload/3883/3883line3.jpg" data-altupdatedat="1787630408000" data-alt="Graph Line 3">) and AMɸ cultured with nanoparticles and labeled with antibodies against all cell surface markers (<img alt="Static equilibrium equation ΣFx=0; diagram illustrating forces balance; physics education tool." src="/files/ftp_upload/3883/3883line2.jpg" data-altupdatedat="1787630408000" data-alt="Graph Line 2">).

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No conflicts of interest declared.

Polyanhydride nanoparticle vaccine platforms have shown efficacy when administered intranasally in single dose regimens5. Measuring the activation of the resident phagocytic cell populations in the lungs induced by this vaccine delivery platform permits evaluation of its potential capability to ultimately promote adaptive immune responses.
	Specifically, harvesting alveolar macrophages from lung lavage fluid and treating them with different formulations of the nanoparticles pr...

<table><tbody><tr><td colspan="4">&lt;strong&gt;cAM Media&lt;/strong&gt;</td></tr><tr><td>DMEM</td><td>Cellgro</td><td>15-013-CV</td><td></td></tr><tr><td>50 mM 2-mercapt&amp;#8211;thanol</td><td>Sigma-Aldrich</td><td>M3148-25ML</td><td></td></tr><tr><td>Penicillin/Streptomycin 10,000 &amp;mu;g/ mL Solution</td><td>Cellgro</td><td>30-002-CI</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;FACS Buffer&lt;/strong&gt;</td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>S671-500</td><td></td></tr><tr><td>Sodium phosphate</td><td>Fisher Scientific</td><td>MK7868500</td><td></td></tr><tr><td>Potassium chloride</td><td>Fisher Scientific</td><td>P217500</td><td></td></tr><tr><td>Potassium phosphate</td><td>Fisher Scientific</td><td>P288-200</td><td></td></tr><tr><td>BSA (Bovine Serum Albumin)</td><td>Sigma-Aldrich</td><td>A7888</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Antibodies&lt;/strong&gt;</td></tr><tr><td>Rat IgG</td><td>Sigma-Aldrich</td><td>I4341</td><td></td></tr><tr><td>Anti-Ms CD16/32</td><td>eBioscience</td><td>16-0161</td><td></td></tr><tr><td>Anti-Ms MHC II haplotype I-A/I-E, clone M5/114.15.2, conjugated to fluorescein isothiocyanate (FITC)</td><td>eBioscience</td><td>11-5321</td><td></td></tr><tr><td>Anti-mouse CD86, clone GL-1, conjugated to allophycocyanin (APC)-Cy7</td><td>Biolegend</td><td>105030</td><td></td></tr><tr><td>Anti-mouse CD40, clone 1C10, conjugated to APC</td><td>eBioscience</td><td>17-0401</td><td></td></tr><tr><td>Anti-mouse CD209, clone 5H10, conjugated to Biotin</td><td>eBioscience</td><td>13-2091</td><td></td></tr><tr><td>Anti-mouse CD11b, clone M1/70, conjugated to Alexa Fluor 700</td><td>eBioscience</td><td>56-0112</td><td></td></tr><tr><td>Anti-mouse F4/80, clone BM8, conjugated to phyc&amp;#8211;rythrin (PE)-Cy7</td><td>eBioscience</td><td>25-4801</td><td></td></tr><tr><td>PE-Texas red conjugated Streptavidin</td><td>BD Biosciences</td><td>551487</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Other Supplies and Reagents&lt;/strong&gt;</td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>A405-20</td><td>Used as 70% (v/v)</td></tr><tr><td>Compressed CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Linweld</td><td>16000060</td><td></td></tr><tr><td>1 mL Syringe</td><td>BD Biosciences</td><td>309659</td><td></td></tr><tr><td>Sovereign 3 &amp;frac12;&quot; Fr Tom Catcatheter</td><td>Kendall</td><td>703021</td><td></td></tr><tr><td>Biosafety Cabinet</td><td>NUAIRE</td><td>Series 22</td><td></td></tr><tr><td>Dissection Scissors</td><td>Fisher Scientific</td><td>138082</td><td></td></tr><tr><td>Forceps</td><td>Roboz Surgical Instruments Co.</td><td>RS-8254</td><td></td></tr><tr><td>PBS, 1X without calcium and magnesium</td><td>Cellgro</td><td>21-040-CM</td><td></td></tr><tr><td>15 mL Centrifuge Tubes with Screw Cap</td><td>VWR international</td><td>21008-216</td><td></td></tr><tr><td>Six-well Tissue Culture Treated Plates</td><td>Costar</td><td>3516</td><td></td></tr><tr><td>Plastic Tube Racks</td><td>Nalge Nunc international</td><td>5970</td><td></td></tr><tr><td>Cell Scraper 24 cm</td><td>Techno Plastic Products</td><td>99002</td><td></td></tr><tr><td>5 mL Polystyrene Round-Bottom Tube</td><td>Falcon BD</td><td>352008</td><td></td></tr><tr><td>Pipet-aid XL</td><td>Drummond Scientific</td><td>4-000-105</td><td></td></tr><tr><td>10, 5, and 2 mL Pipettes</td><td>Fisher Scientific</td><td>13-675</td><td></td></tr><tr><td>200 and 10 &amp;mu;L micropipettors</td><td>Gilson Pipetman</td><td>F123601</td><td></td></tr><tr><td>200 and 10 &amp;mu;L pipette tips</td><td>Fisher Scientific</td><td>02-707</td><td></td></tr><tr><td>BD Stabilizing Fixative</td><td>BD Biosciences</td><td>338036</td><td></td></tr><tr><td>Isoton II Diluent</td><td>Beckman Coulter Inc.</td><td>8546719</td><td></td></tr><tr><td>Zap-oglobin II Lytic Reagent</td><td>Beckman Coulter Inc.</td><td>7546138</td><td></td></tr><tr><td>Coulter Counter Polystyrene Vials</td><td>Beckman Coulter Inc.</td><td>14310-684</td><td></td></tr><tr><td>Test Tubes</td><td>BD Biosciences</td><td>352008</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Refrigerated Centrifuge</td><td>Labnet International</td><td>50075040</td><td></td></tr><tr><td>Humidified Incubator CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Nuaire</td><td>Model Autoflow 8500</td><td></td></tr><tr><td>FACSCanto Flow Cytometer</td><td>BD Biosciences</td><td>338960</td><td></td></tr><tr><td>Coulter Particle Counter Z1</td><td>Beckman Coulter Inc.</td><td>WS-Z1DUALPC</td><td></td></tr><tr><td>Sonicator Liquid Processing Equipment with Microtip</td><td>Misonix</td><td>Model No. S-4000</td><td></td></tr></tbody></table>

harvesting murine alveolar macrophages and evaluating cellular activation induced by polyanhydride nanoparticles

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

The overall goal of this procedure is to evaluate the ability of poly anhydride nanoparticles to activate purified mirroring alveolar macrophages. First, the trachea of the mouse is surgically exposed and along lavage is performed to harvest cells from the respiratory tract. Alveolar macrophages are then spun down to isolate them and cultured overnight.A poly anhydride nanoparticle suspension is then prepared and added to the alveolar macrophages. The macrophages are incubated to allow for activation after 48 hours. Cell surface marker expression is analyzed by flow cytometry.Analysis of the data enables identification of formulations of Polly Anhydride nanoparticles capable of activating mirroring alveolar macrophages. This metal can help to answer questions in the fields of biomaterials and immunology. Specifically, we can identify biomaterials with intrinsic adjuvant activity as evidenced by the ability to activate the engine presenting cells, including alveolar macrophages.We first had the idea to use this method in our research when we discovered that a poly anhydride nano vaccine platform against respiratory pathogens provided complete protection against bacterial pneumonia. In this present work, we sought to investigate the specific interactions of the vaccine platform with respiratory immune cells. Begin this procedure by placing a euthanized mouse on its back.Spray the mouse with 70%ethanol, although a six to eight week old C 57 BL six mouse is used here, this technique can be performed on any strain of mouse. Locate the sternum and at a 30 degree angle from the abdomen, insert a 23 gauge needle with a one milliliter syringe. About 50 millimeters into the thoracic cavity, collect 0.4 to one milliliter of blood Generally in the wheels.Performing this procedure will struggle getting an off sale for their experiments. That's why it's really important to perform carefully as demonstrated here. To perform the lavage, turn the mouse onto its abdomen and spray it with 70%ethanol.Then pinch the skin about halfway down the spine using dissection scissors that have been sterilized with 70%ethanol. Make a small incision through the skin. Grab the skin on both sides of the incision and pull in opposite directions along the cranial coddle axis of the mouse.Once the muscles and glands of the neck are exposed, carefully cut muscle tissue. Then using forceps. Pull the tissue toward the anterior of the mouse to expose the larynx, trachea, and esophagus.Avoid cutting the jugular vein or carotid artery. The trachea is identifiable as the tissue with cartilaginous rings around it. With the forceps delicately, grasp the trachea and gently lift and separate it from the esophagus, which is located on the dorsal side.Fill a one cc syringe fitted with a tomcat catheter with sterile PBS. Next at a 45 degree angle. Make a small incision in the trachea anterior to the forceps, but posterior to the larynx.Avoid completely transecting the trachea, which will prevent it from retracting into the body cavity. Using forceps, hold the trachea and gently insert the catheter about 20 millimeters into the trachea incision site. Then grip the trachea that is around the catheter tube to free the other hand while externally massaging the chest.Use a one milliliter syringe fitted with the catheter to gently infuse 0.75 milliliters of room temperature sterile PBS into the lungs. Then gently aspirate the PBS back into the syringe. Repeat this process three times.Disconnect the syringe from the catheter and dispense the lavage fluid into a 15 milliliter centrifuge tube. Fill the syringe with 0.75 milliliters of fresh PBS and reconnect the syringe to the catheter and repeat the lavage. Fluid recovery may be partial, so less volume may be obtained in the volume infused.If cells will be collected from more than one mouse. Place the 15 milliliter tube with the harvested cells on ice until all lung lavages have been performed. To enrich the population of alveolar macrophages in the harvested cells, centrifuge the lung lavage fluid at 250 times G for 10 minutes at four degrees Celsius.Using sterile technique, decant the supernatant into an appropriate waste container Inside a biosafety cabinet. Resus suspend the cell pellet by gently rigging the centrifuge tube across the top of a test tube rack. Add one milliliter of complete alveolar macrophage medium to resuspend the cells to enumerate the cells.Pipette 20 microliters of cells into 10 milliliters of isotone two diluent in a culture counter vial. Add three to four drops of zog globin, two lytic reagent into the vial and gently inverted three to four times. Then load the sample vial into a colter particle counter Z one counter.Be sure to program the counter with the dilution factor of two times 10 to the fourth to display the cell concentration as a number of cells per milliliter. Once the cell concentration has been determined, dilute the cells to 2.5 times 10 to the fifth per milliliter with complete alveolar macrophage medium in a 15 milliliter tube. Then dispense two milliliters of the solution into each well of a six well tissue culture dish.To enrich the alveolar macrophages, incubate the culture dishes in a 37 degrees Celsius humidified incubator with a 5%CO2 atmosphere for six hours. During the incubation, the macrophages will adhere to the bottom of the well following the incubation, gently aspirate the supernatant taking care not to disrupt the adherent cells. Then rinse the wells using sterile calcium and magnesium free PBS.To remove non-adherent cells and debris, add two milliliters of complete alveolar macrophage medium to each. Well then incubate the cells at 37 degrees Celsius overnight. After this enrichment step, approximately 87%of the cells will be positive for the macrophage marker CD 11 B and F four 80.Once the alveolar macrophage population has been enriched, the next step of the procedure is to add poly anhydride nanoparticles first fabricate nanoparticles via poly anhydride antis solvent nano encapsulation. In this process, the polymer is dissolved in methylene chloride and precipitated in pentane. Weigh out the appropriate amount of poly anhydride nanoparticles using sterilized whey paper.Here, 125 micrograms of particles are added for each milliliter of culture. Add the particles to a 1.5 milliliter micro centrifuge tube, resuspend the nanoparticles in the appropriate amount of ice cold, complete alveolar macrophage medium, and place it on ice. Next, you sterilize the microtip of a benchtop sonicate.Spray it with 70%ethanol and wipe with a sterile kim wipe while keeping the tube on ice. Sonicate at 10 to 20 joules for 30 to 45 seconds. Observe the nanoparticle suspension macroscopically if the particles are not sufficiently dispersed.Place the suspension on ice for one to two minutes to ensure that the nanoparticles are below the glass transition temperature of the polymer. Then sonicate again for 30 to 45 seconds. The intent of the sonication step is to adequately disperse the nanoparticles as the 50 50 CCP tag.CPH nanoparticles tend to aggregate when exposed to an aqueous environment. However, some small aggregates of nanoparticles may still remain in the suspension. The nanoparticle suspension should be kept on ice and used immediately to prevent premature surface erosion or aggregation.Remove the culture dishes containing the alveolar macrophages from the incubator and place them in the biosafety cabinet. Tilt the plate taking care not to disturb the A adherent cells and pipette off the amount of medium that will be added to the nanoparticle suspension Vortex. The poly anhydride nanoparticle suspension briefly then pipette up to 0.3 milliliters of the nanoparticle suspension into the appropriate wells.Again, taking care not to disturb the adherent cells. The particles will settle to the bottom of the well and contact the cells. Proceed with subsequent wells and desired treatment groups.Include positive and negative control groups such as medium only, and 200 nanograms per milliliter. Lipopolysaccharide to designated wells, then incubate at 37 degrees Celsius for 48 hours. Following the incubation, the alveolar activation can be assessed by a flow cytometry as described in the accompanying document.Alveolar macrophages were obtained from C 57 BL six mice and enriched as described in the video article to assess cellular activation. 125 micrograms per milliliter 50 50 CPT CPH poly anhydride nanoparticles were added to five times 10 to the fifth cells per milliliter of enriched alveolar macrophages for 48 hours. Expression of the alveolar macrophage markers, MHC two, CD 40, CD 86, and sire were then assessed by antibody staining and flow cytometry as can be seen in this histogram.Alveolar macrophages incubated with nanoparticles indicated by the solid blue line, increased surface expression of MHC two, a molecule required for the presentation of antigen to CD four T cells as compared to those not treated with nanoparticles as expected. Lower levels of staining were seen in the fluorescence minus one FMO controls, indicating that the observed signal is not due to autofluorescence alveolar macrophages incubated with nanoparticles indicated by the solid blue line enhanced expression of CD 40. A co-stimulatory molecule necessary for appropriate activation of T cells as compared to untreated cells.Expression of another T-cell co-stimulatory molecule CD 86 was also increased following treatment with poly anhydride nanoparticles for 48 hours. The expression of CY a C type lectin receptor that binds carbohydrate motifs present on the surface of pathogens is greatly enhanced on alveolar macrophages after poly anhy nanoparticle treatment. After watching this video, you should have a good understanding of how to perform along lavash with the purpose to harvest marine vir macrophages, how to culture those cells, and also how to stimulate those cells to measure or evaluate the cellular activation of those cells with poly hydro nanoparticles.While attempting this procedure, it is important to use sterile laboratory techniques to prevent contamination of the primary cells being harvested and to prevent introduction of contaminants into the treatments.

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Learn to harvest murine alveolar macrophages via bronchoalveolar lavage and evaluate their activation by polyanhydride nanoparticles using flow cytometry. This protocol enables immunology and nanotechnology research.

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

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