Name: analyses of proteinuria, renal infiltration of leukocytes, and renal deposition of proteins in lupus-prone mrl/lpr mice
Uploaded: 2022-06-08T13:00:00
Description: Watch this Scientific Journal Video about Analyses of Proteinuria, Renal Infiltration of Leukocytes, and Renal Deposition of Proteins in Lupus-prone MRL/lpr Mice at JoVE.com

We thank the Flow Cytometry Core Facility, the Histopathology Laboratory, the Fralin Imaging Center at Virginia Polytechnic Institute, and State University for technical support. This work is supported by various NIH and internal grants.

The present protocol is approved by the Institutional Animal Care and Use Committee (IACUC) at Virginia Tech. Since lupus disease has a higher incidence in females, only female MRL/lpr mice were used. The sample collection was started at 4 weeks of age and finished at 15 weeks. The mice were obtained from commercial sources (see Table of Materials) and were bred and maintained in a specific pathogen-free environment following the institutional guidelines.
1. Proteinuria ...

<ol>
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LN is a leading cause of mortality in SLE patients, and factors aggravating the disease remain unclear. The application of this protocol is to characterize renal function using multiple methods, including measurement of proteinuria, FACS analysis of isolated kidney leukocytes, and immunofluorescence staining of frozen kidney sections.
One important point to consider while collecting urine is that one has to be consistent with the time of the day and the location of urine collection. Mice are n...

The kidney&#39;s primary function is the elimination of toxic substances through urine while maintaining the homeostasis of water and salts1. This function is threatened in patients with systemic lupus erythematosus (SLE), leading to so-called lupus nephritis (LN). LN is a consequence of the immune system attacking the kidney, leading to persistent kidney inflammation, therefore losing its ability to clean waste from the blood and regulate salt and fluid concentrations. This will eventually lead to renal failure, which can be fatal. During the nephritic process, circulating B cells, T cells, and monocytes are recruited to the kidney, secreting chemokines, cytokines, and immune complex-forming autoantibodies. This ultimately results in endothelial cell damage, membranous injuries, renal tubular atrophy, and fibrosis2.
MRL/Mp-Faslpr (MRL/lpr) lupus-prone mice is a classical mouse model exhibiting lupus-like clinical signs that resemble human SLE3. This model has been instrumental in understanding one of the leading causes of mortality in SLE patients, lupus nephritis (LN)4. In both human and mouse SLE, LN is characterized by gradual inflammation triggered by renal deposition of immune complexes, followed by complement activation, recruitment of inflammatory cells, and loss of renal function5. The immune complex deposition is the first step to induce chemokine and cytokine production by intrinsic renal cells, which expands the inflammatory response by recruiting immune cells6. The current protocol presents several techniques to follow renal disease progression that analyze cell infiltration and immune complex deposition.
Urine collected every week allows for detection and visualization of the time course of proteinuria before, during, and after lupus onset. Proteinuria as a biomarker can determine the biological progression of LN. Other advantages of this technique are that it is non-invasive, cost-efficient, and easy to implement7. When the kidney is working perfectly, the proteinuria level is consistently low; however, in MRL/lpr mice, after 8-9 weeks of age, a gradual increase of the proteinuria level, that is eventually high enough to cause renal failure8, is observed. Multiple reagent strips and colorimetric reagents are commercially available to monitor the issue. However, the Bradford assay is cheap and very accurate in determining the onset of proteinuria and the course of lupus nephritis. This assay is quick, and the reagent is not affected by the presence of solvents, buffers, reducing agents, and metal-chelating agents that may be in your sample9,10,11.
One important aspect to consider is cell infiltration in the kidney. These infiltrates promote pathogenesis by triggering the secretion of soluble factors such as cytokines to worsen inflammation12. To better understand what cell populations are present in the infiltrates, a useful method is to isolate leukocytes13. Here, the detection of renal infiltration of B cells is used as an example. The procedure begins with a digestion process with deoxyribonuclease (DNase) and collagenase, followed by density gradient separation that removes debris, red blood cells, and dense granulocytes. The reason for isolating B cells (CD19+) and plasma cells (CD138+) is that lupus kidneys can concentrate these cells14. It is suggested that the presence of B cells in small aggregates in the kidney can indicate clonal expansion and, consequently, immunoglobulin (Ig) production. Plasma cells are well-known to be present in these aggregates as well15. Once leukocytes have been isolated, fluorescence-activated cell sorting (FACS) can be used to analyze the cells of interest upon staining with different fluorescence-conjugated antibodies.
Immunofluorescence is one of the immunohistochemistry (IHC) detection methods that allows for fluorescent visualization of proteins in 4 &#956;m thick kidney tissue samples. Other IHC detection methods depend on the nature of analytes, binding chemistry, and other factors16. Immunofluorescence is a rapid identification method that exposes the antigen to its counterpart antibody labeled with a specific fluorochrome (or a fluorescent dye). When excited, it produces light that a fluorescence microscope can detect. This technique can be used to observe the deposition of complement C3 and IgG2a17. Excessive complement cascade activation could be associated with an uncontrolled immune response and loss-of-function18. Immune deposition of anti-double-stranded DNA (anti-dsDNA) autoantibodies in the kidney is a major concern19, where those with IgG2a isotype have been associated with LN20. Specifically, anti-dsDNA antibodies exhibit more pathogenicity and affinity to nuclear materials, forming immune complexes21. When IgG2a is present, the complement cascade, including C3, is activated to clear the immune complexes22. The C3 and IgG2a markers can be quantified individually or overlaid to establish their correlation.
Notably, serum creatinine measurement is another reliable technique that can be used together with microscopic hematuria and kidney biopsies to diagnose LN23. However, the presence of proteinuria is a strong indicator of glomerular damage. In that sense, monitoring the proteinuria level during lupus can detect disease onset and complement other methods for diagnosing lupus. In addition, immune complexes deposited in glomeruli can induce an inflammatory response, activate the complement system, and recruit more inflammatory cells. Another noteworthy point of this protocol is B cell infiltration in the kidney. This, together with the infiltrated T cells, amplifies local immune responses that trigger organ damage. Importantly, the classification of LN is not only based on glomerular morphologic changes seen in microscopy but also immune deposits observed with immunofluorescence. Therefore, in this protocol, accurate and cost-effective methods for the analysis of renal function are offered in laboratory settings.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x Tris-Buffered Saline (TBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>J60764.K2</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Human/Mouse C3</td>
			<td>Cedarlane</td>
			<td>CL7632F</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD138 BV711</td>
			<td>Biolegend</td>
			<td>142519</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD45 AF700</td>
			<td>Biolegend</td>
			<td>103127</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9418-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Sigma-Aldrich</td>
			<td>11088882001</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope LSM 880</td>
			<td>Zeiss</td>
			<td>LSM 880</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin jar</td>
			<td>Fisher Scientific</td>
			<td>50-212-281</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryomold</td>
			<td>Fisher Scientific</td>
			<td>NC9511236</td>
			<td></td>
		</tr>
		<tr>
			<td>Density gradient medium</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td>Percoll</td>
		</tr>
		<tr>
			<td>DEPC-Treated water</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9906</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma-Aldrich</td>
			<td>D4527</td>
			<td></td>
		</tr>
		<tr>
			<td>dsDNA-EC</td>
			<td>InvivoGen</td>
			<td>tlrl-ecdna</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic Acid</td>
			<td>Fisher Scientific</td>
			<td>S311-500</td>
			<td>EDTA</td>
		</tr>
		<tr>
			<td>EVOS M5000 Microscope imaging system</td>
			<td>Thermo Fisher Scientific</td>
			<td>AMF5000</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Fusion Cell sorter</td>
			<td>BD Biosciences</td>
			<td>FACS Fusion</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum - Premium, Heat Inactivated</td>
			<td>R&#38;D systems</td>
			<td>S11150H</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand 96-Well Polystyrene Plates</td>
			<td>Fisher Scientific</td>
			<td>12-565-501</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad prism</td>
			<td>GraphPad</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>14175-079</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>National Institutes of Health</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactobacillus reuteri Kandler et al.</td>
			<td>ATCC</td>
			<td>23272</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM non-essential amino acids</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>MRL/MpJ-Fas lpr /J Mice (MRL/lpr)</td>
			<td>Jackson Lab</td>
			<td>485</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail enamel</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any conventional store</td>
		</tr>
		<tr>
			<td>O.C.T compound</td>
			<td>Tisse-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen</td>
			<td>Sigma-Aldrich</td>
			<td>Z377821</td>
			<td></td>
		</tr>
		<tr>
			<td>Peel-A-Way Disposable Embedding Molds</td>
			<td>Polysciences</td>
			<td>R-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>70011069</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce 20x TBS Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>28358</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Coomassie Plus (Bradford) Assay kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23236</td>
			<td>Albumin standard included</td>
		</tr>
		<tr>
			<td>ProLon Gold Antifade Mountant</td>
			<td>ThermoFisher</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Rat Anti-Mouse CD16/CD32</td>
			<td>BD Biosciences</td>
			<td>553141</td>
			<td>FcR block</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax M5</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td>SoftMax Pro 6.1 software</td>
		</tr>
		<tr>
			<td>Sterile cell Strainers 100 &#181;M</td>
			<td>Fisher Scientific</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vancomycin Hydrochloride</td>
			<td>Goldbio</td>
			<td>V-200-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Zombie Aqua</td>
			<td>Biolegend</td>
			<td>423102</td>
			<td>fluorescent dye for flow cytometry analysis</td>
		</tr>
	</tbody>
</table>

analyses of proteinuria, renal infiltration of leukocytes, and renal deposition of proteins in lupus-prone mrl/lpr mice

Systemic lupus erythematosus (SLE) is an autoimmune disorder with no known cure and is characterized by persistent inflammation in many organs, including the kidneys. Under such circumstances, the kidney loses its ability to clean waste from the blood and regulate salt and fluid concentrations, eventually leading to renal failure. Women, particularly those of childbearing age, are diagnosed nine times more often than men. Kidney disease is the leading cause of mortality in SLE patients. The present protocol describes a quick and simple method to measure excreted protein levels in collected urine, tracking lupus progression over time. In addition, an approach to isolate kidney mononuclear cells is provided based on size and density selection to investigate renal infiltration of leukocytes. Furthermore, an immunohistochemical method has been developed to characterize protein deposition in the glomeruli and leukocyte infiltration in the tubulointerstitial space. Together, these methods can help investigate the progression of chronic inflammation associated with the kidneys of lupus-prone MRL/lpr mice.

The protocol uses multiple methods to assess MRL/lpr mice for lupus nephritis. First, a procedure is described to study increased proteinuria levels due to kidney dysfunction over time. As shown in Figure 1, female mice were treated with oral gavage of 200 &#181;L of phosphate-buffered saline (1x PBS) as the control group and probiotic Lactobacillus reuteri as the treatment group, at a concentration of 109 cfu/mL, twice a week. Treatment started at 3 weeks old and finishe...

Watch this Scientific Journal Video about Analyses of Proteinuria, Renal Infiltration of Leukocytes, and Renal Deposition of Proteins in Lupus-prone MRL/lpr Mice at JoVE.com

Analyses of Proteinuria, Renal Infiltration of Leukocytes, and Renal Deposition of Proteins in Lupus-prone MRL/lpr Mice

The present protocol describes a method to track lupus progression in mice. Two additional procedures are presented to characterize lupus nephritis based on cell infiltration and protein deposition in the kidneys.

Systemic lupus erythematosus (SLE) is an autoimmune disorder with no known cure and is characterized by persistent inflammation in many organs, including ...

These methods can help investigate the progression of chronic inflammation associated with kidneys in lupus patients. We are offering a reliable and cost effective alternative that can be used to monitor the disease course in lupus. Begin sterilizing the hood by spraying 70%ethanol and place a sterile plastic sheet on the surface.Then prepare tips, 1.5 milliliter tubes and a pipette to collect about 20 microliters of the urine. Take the cage and spray 70%ethanol before placing it inside the hood. Afterward, hold the mouse with one hand and use the other hand to gently massage the ventral area from top to bottom.Using a 1.5 milliliter tube or pipette, collect the urine directly. Or if the sample drops collect it from the plastic sheet. Then put the mouse back in the cage.Cut the dissected kidneys into grain size and digest in five milliliters of digestion buffer containing Collagenase and DNase 1 in RPMI 1640 medium containing HEPES for one hour with continuous gentle shaking at 37 degrees Celsius. Add 10 milliliters of ice cold PBS containing 10 millimolar of EDTA and incubate for 10 minutes on ice. Then vortex the suspension twice.Later, filter the cell suspension through a 100 micrometer strainer and wash with 10 milliliters of HBSS full. Afterward, centrifuge at 350 times G for 10 minutes at room temperature. Prepare a 30, 37 and 70%stock isotonic Percoll solution and add a layer of 37%Percoll solution on top of the 70%solution.Re suspend the cells in five milliliters of 30%stock isotonic Percoll. Using a Pasteur pipette slowly load 37%of five milliliters top and 70%of five milliliters on the bottom, making stock isotonic Percoll gradient. Then, centrifuge with up number nine and down number zero at 1000 times G for 30 minutes at room temperature.Afterward, collect leukocytes from the 37 to 70%interface and re suspend them in five milliliters of C 10. Again, centrifuge at 350 times G for 10 minutes at four degrees Celsius. Subsequently re suspend the cell pellet in three milliliters of fax buffer and centrifuge at 350 times G for five minutes at four degrees Celsius.Then discard the supernatant leaving about 50 microliters of the total volume. Add 50 microliters of FC receptor block per tube. Following this mix and incubate on ice in the dark for 10 minutes.Later, add two milliliters of fax buffer for washing. Then, centrifuge at 350 times G for five minutes at four degrees Celsius and discard the supernatent leaving about 50 microliters of the total volume. Next, add 20 microliters of the appropriate fluorescent dye and let it stand for 15 to 30 minutes on ice.Add 50 microliters of antibody mix per tube containing CD 138 brilliant violet seven 11 and CD 45 Alexa Fluor 700 and incubate on ice in the dark for 15 to 30 minutes. Afterward, add three milliliters of fax buffer. Centrifuge at 350 times G for five minutes at four degrees Celsius.Remove the supernatant by vacuum leaving about 50 microliters of the total volume. Later, re suspend the cell pellet in 200 microliters of PBS. To collect the sample embed the half kidney in the small plastic cryomold with the optimal cutting temperature compound.Then add dry ice into a polystyrene foam box and carefully place the cryomold on top of the dry ice. Next, remove the cryomold, wrap it in aluminum foil and store it at minus 80 degrees Celsius until further use. Then fix the dry to four micrometer sections with cold acetone for 10 minutes.After fixing the slides, air dry for 0.5 to one hour and store them at minus 20 degrees Celsius for a short time or minus 80 degrees Celsius for a long time. To stain the samples, refix the slides in cold acetone for five to 10 minutes and allow the sections to warm up to room temperature. Afterward, using a pat pen, draw a hydrophobic circle around the section and allow it to dry.Then place slides in TBS in the Copeland jar for 20 minutes and shake gently by putting the jar on the shaker. Subsequently, pour off TBS and fill the jar with TBS 5%BSA and 0.1%TW 20. Later, incubate the jar for 20 minutes by shaking gently on the shaker.Then, take the slides out and wipe the bottom of the slides. Add 100 microliters of conjugated antibodies C3 fit C and IgG2 APE to the section and incubate room temperature in a humidified chamber for one hour. Wash the section thrice in PBS 5%BSA and 0.1%TW 20 for 10 minutes on the shaker and then shake dry the slides.Mount the section with an anti-fade reagent and then with a cover slip. Then store the slides at four degrees Celsius until viewing under the microscope. For image analysis using the image J software, outline the desired region.Afterward, set standard parameters by clicking on analyze then set measurements and measure area to get the results. Next, transfer the data obtained from the measure windows to a spreadsheet. Once done, click on analyze to measure the region and transfer the data again to the previous spreadsheet.The protein levels in the urine of MRL LPR mice were detected over time where they were treated with phosphate buffered saline as the control group and lactobacillus reuteri as the treatment group. The mice group treated with L.reuteri at a more aggressive proteinuria progression than the control group. The fax analysis was performed for the isolated kidney leukocytes to analyze the plasma cells.Further, the mice were treated with vancomycin or vancomycin along with Escherichia coli double stranded DNA. Although Escherichia coli double stranded DNA was expected to trigger the renal infiltration, no significant difference was found in the percentage of plasma cells. The complement C3 and IgG2a were detected through immunofluorescence staining on female MRL IPR kidney sections.The effective environmental factors on the renal deposition of C3 and IgG2a was compared for the in-house mice bread in the animal facility and those purchased from a vendor. The immunohistochemical analysis did not show any difference between the two groups. Adding each layer of the percentage stock ISO 20 Percoll solution can be challenging, and if they mix you have to start over again.This procedure helps with the flow cytometer and in vitter experiments, so you cannot study the different kidney cell type of populations. This procedure allows us to study the B-cells infiltrates in the kidneys and there is not a lot of information in the literature.

analyses-proteinuria-renal-infiltration-leukocytes-renal-deposition

Research

JoVE Journal

Immunology and Infection

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Renal Subcapsular Transplantation of 2'-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation is an important method for investigating thymic epithelial cell function and T cells maturation in vivo. Here we will describe the experimental methods used within our laboratory to transplant 2&#8217;-deoxyguanosine (to deplete donor&#8217;s lymphocytes) treated embryonic thymus into the renal capsule of an athymic nude mouse. This method is both simple and efficient and does not require special skills or devices. The results obtained via this simple method showed that transplanted thymus can effectively support the recipient&#8217;s T cells production. Additionally, several key points with regards to the protocol will be further elucidated.

We provide a simple and efficient method to transplant 2&#8217;-deoxyguanosine treated E18.5 thymus into the renal capsule of a nude mouse. This method should aide in the study of both thymic epithelial cells function and T cells maturation.

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation ...