This work was supported by grants #1K08HL088260 and #1R01HL133462-01A1 (NHLBI) (A.B.P., H.N., S.J.), and the Batchelor Foundation for Pediatric Research (D.M., H.N., S.J., A.A.H., A.B.P.). C57BL/6 and BALB/c mice used in this study were either bred in our facility or provided by Jackson Labs or Taconic.

All studies were conducted under rodent research protocols reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Miami Miller School of Medicine, which meets the veterinary standards set by the American Association for Laboratory Animal Science (AALAS).
1. Preparation of Solutions
<ol>
	<li>As described in Table 1, prepare the Colon Buffer, Silica-Based Density Separation Media 100%, Silica-Based Density Separation Media 66%, Silica-based Density Separation Media 44%, Collagenase E Digestion Buffer, and FACS Buffer.
	<ol>
		<li>Prepare Colon Buffer the day prior to the procedure and store overnight at 4 &#176;C.</li>
		<li>Prepare 100% Silica-Based Density Separation Media the day prior to the procedure and stored overnight at 4 &#176;C, placing at room temperature the morning of the procedure to thaw.</li>
		<li>Prepare 66% and 44% Silica-Based Separation Media in the morning of the isolation, using room temperature 100% Silica-Based Density Separation Media and Colon Buffer.</li>
		<li>Measure the appropriate amount of Clostridium histolyticum-derived Collagenase E and store at -20 &#176;C overnight prior to the procedure. The following morning, dissolve in the appropriate volume of Colon Buffer to derive Collagenase Digestion Buffer. For all incubations with Collagenase Digestion Buffer on day of the procedure, pre-warm the solutions to 37 &#176;C.</li>
	</ol>
	</li>
	<li>For the entire of the protocol steps, keep one centrifuge at 20 &#176;C and the rotation speed of 859 x g, with brakes inactivated (0 deceleration) for gradient centrifugation. Set another to 4 &#176;C and rotation speed of 859 x g, with the standard deceleration, for wash steps.</li>
</ol>
2. Harvesting the Colon
<ol>
	<li>Euthanize the mouse via CO2 asphyxiation followed by AALAC-approved confirmatory method.</li>
	<li>Place the mouse in a supine position and spray the fur with 70% ethanol. Using large tissue scissors, make a vertical midline incision and expose the intact peritoneum.</li>
	<li>Using fine dissection scissors, open the peritoneum. Use forceps to move the small bowel to one side and expose the descending colon. Slightly pull upward on the descending colon to maximally expose the rectal portion of the colon. Cut the distal rectum deep in the pelvis and dissect and remove the entire colon as one unit, from the distal rectum to cecal cap.</li>
	<li>Transfer the colon in 20 mL chilled Colon Buffer in a 50 mL polypropylene tube.</li>
</ol>
3. Cleaning the Colon
<ol>
	<li>Place the colon on a moistened paper towel and extract the solid stool by applying mild pressure to the bowel wall with the blunt end of scissors or forceps.</li>
	<li>Place the colon in a Petri dish and flush the gut with 10 mL of chilled Colon Buffer using a 10 mL syringe with 18 G blunt fill needle.</li>
	<li>Transfer the colon to a Colon Buffer moistened paper towel and remove the mesentery and fat with the sharp end of scissors.</li>
	<li>Place the colon in a Petri dish filled with 5-10 mL chilled Colon Buffer agitating manually to wash remaining colonic contents. Repeat 2-3 times.</li>
	<li>Cut the colon longitudinally from its more muscular rectal end to the proximal colon (generating a single rectangular open colon piece) in a Petri dish filled with fresh chilled Colon Buffer. Discard existing media and refill with clean chilled Colon Buffer.</li>
	<li>Wash the intestine 3 times by vigorously swirling it in the Petri dish and replacing the 5-10 mL of chilled Colon Buffer after with each wash.</li>
	<li>Place the rectangular colon tissue on a paper towel moistened with Colon Buffer and cut it by slicing it horizontally and then into small fragments (3 mm x 3 mm sections).</li>
	<li>Collect the colon fragments carefully using fine forceps into 20 mL chilled Colon Buffer in a 50 mL polypropylene conical tube. </li>
	<li>Wash the colon fragments 3 times, each wash in 20 mL Colon Buffer, by vigorously swirling the tube for 30 s. Between each agitation, allow the tissue fragments to settle to the bottom of the tube. Decant or vacuum aspirate the supernatant while preventing tissue fragment loss in the aspiration process between each wash. 
	NOTE: There is no need to change the tube after each wash.</li>
</ol>
4. Collagenase Digestion 1
<ol>
	<li>Add 20 mL of the Collagenase Digestion Buffer to the washed colon fragments in the 50 mL polypropylene conical tube.</li>
	<li>Place the closed 50 mL tube at 37 &#176;C in an incubated orbital shaker with the rotation rate set at 2 x g for 60 min. Ensure the tissue fragments are in constant motion during agitation; if necessary, increase the rotation rate incrementally to ensure that no tissue fragments settle to the tube bottom.</li>
</ol>
5. Prepare Silica-based Separation Media Gradients
<ol>
	<li>Prepare 66% and 44% Silica-based Density Separation Media, using 100% Silica-based Density Separation Media at 20 &#176;C (room temperature) and Colon Buffer.</li>
	<li>Pour 5 mL of 66% Silica-based Density Separation Media into each of 3 separate 15 mL polypropylene tubes. Prepare 3 tubes per colon. This forms the higher density base of the gradient isolation procedure, onto which lower density separation media will be layered to create the separation gradient.</li>
	<li>Store at 20 &#176;C until use.</li>
</ol>
6. Collection of Supernatant from Digestion 1
<ol>
	<li>Collect only the supernatant using a 25 mL serological pipette and filter the supernatant through a 40 &#956;m pore filtration fabric cell strainer placed into a clean 50 mL polypropylene conical tube, after Collagenase Digestion 1 is completed. Be careful not to aspirate any existing tissue fragments. 
	NOTE: Retain any remaining visible tissue fragments in the tube. These will undergo second collagenase digestion (step 8).</li>
</ol>
7. Quenching Collagenase Digestion Buffer
<ol>
	<li>Fill the 50 mL polypropylene tube completely with chilled Colon Buffer. 
	NOTE: Collagenase is active at 37 &#176;C; hence chilled buffer inactivates this enzyme.</li>
	<li>Centrifuge the tube at 4 &#176;C at 800 x g for 5 min.
	<ol>
		<li>Discard the supernatant via vacuum aspiration. Wash cells with 25 mL of fresh Colon Buffer and centrifuge at 800 x g for 5 min.</li>
		<li>Resuspend the pellet in less than 1 mL of fresh chilled Colon Buffer.</li>
		<li>Place the 50 mL polypropylene conical tube on ice.</li>
	</ol>
	</li>
</ol>
8. Collagenase Digestion 2
<ol>
	<li>Repeat step 4 (Digestion 1) with the remaining tissue fragments retained from step 6.1.</li>
</ol>
9. Tissue Disaggregation Following Digestion 2
<ol>
	<li>Flush the tissue fragments vigorously back and forth between the tube and a 10 mL syringe through an 18 G blunt-end needle.</li>
	<li>Repeat this flush for a minimum of 7-8 complete passages, continuing until no gross tissue fragments or debris are visible.</li>
</ol>
10. Filter Cells
<ol>
	<li>Pass the tissue disaggregation suspension through a 40 &#956;m-pore filtration fabric cell strainer into a clean 50 mL polypropylene tube.</li>
	<li>Wash the filtration fabric cell strainer with 10 mL chilled Colon Buffer to recover any cells ensnared in the filter.</li>
</ol>
11. Quenching Collagenase Digestion
<ol>
	<li>Fill the 50 mL polypropylene conical tube to the rim with chilled Colon Buffer. 
	NOTE: The temperature of Colon Buffer is critical to ensure quenching of collagenase activity.</li>
	<li>Spin at 4 &#176;C and 800 x g for 5 min.</li>
	<li>Discard the supernatant via vacuum aspiration.</li>
	<li>Wash by resuspending in 25 mL of fresh chilled Colon Buffer, followed by centrifugation at 4 &#176;C, 800 x g for 5 min.</li>
	<li>Discard the supernatant via vacuum aspiration.</li>
	<li>Pool the resuspended pellet from Collagenase Digestion 1 (step 7) to its corresponding tube from step 11.4.</li>
	<li>Repeat Step 11.4 (wash and centrifugation).</li>
</ol>
12. Silica-based Density Separation Media Gradient Separation
NOTE: Perform steps 12-18 as quickly as possible, to ensure rapid quenching of collagenase activity.
<ol>
	<li>Following step 11.7, resuspend each pellet in 24 mL total of 44% Silica-based Density Separation Media per colon.</li>
	<li>Slowly layer 8 mL of the media from step 12.1 onto each of three tubes prepared at step 5.2 (containing 66% Silica-based Density Separation Media), using a 10 mL serological pipette. Maintain a steady and slow flow of the 44% Density Separation Media while layering the gradient in order to avoid disruption of the interface.</li>
	<li>Carefully balance all tubes within the centrifuge buckets using a weigh scale or a balance.</li>
	<li>Spin the tubes 20 min at 859 x g in a centrifuge without brake at 20 &#176;C. Allow the rotors to come to complete rest before removing tubes, taking care not to disrupt the cells at the gradient interface.</li>
</ol>
13. Collect Mononuclear Cells from the Gradient Interface
<ol>
	<li>Visualize the gradient interface (near the 5 mL mark), where typically a 1-2 mm thick white band (containing MNC) is present. 
	NOTE: One may or may not see a white band. However, MNC will be at this interface and should cloud the clarity of the gradient interface.</li>
	<li>Vacuum aspirate and discard the top 7 mL of the top gradient to allow easier pipette access to the interface.</li>
	<li>Using continuous manual suction and steady rotating wrist motion, collect the interface layer of cells into a clean 50 mL polypropylene conical tube. Collect until the interface between the 2 gradients is clear and refractile (clear of cells).</li>
	<li>Fill the collection tube with 50 mL of chilled FACS Buffer. Spin at 4 &#176;C, 800 x g for 5 min.</li>
	<li>Aspirate the supernatant via vacuum aspiration and resuspend the pellet in 1 mL of FACS Buffer.</li>
	<li>Count the cells on a hemocytometer at a 1:2 dilution using appropriate dead cell exclusion methods.</li>
	<li>Proceed to FACS staining or other assays with freshly isolated colonic MNC.</li>
</ol>

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The authors declare no competing financial interests.

This visual protocol describes well-tolerated methods for the isolation of colonic mononuclear cells including lamina propria lymphocytes (LPL). Given that this protocol was optimized in evaluating severe post-transplant mouse colitis models where inflammatory cytokines and tissue injury lend themselves to poor viability of recovered MNC, we anticipate that these methods can be translated to other applications requiring phenotypic analysis of colonic MNC. These include but, are not limited to assessing colon inflammation...

Though the gastrointestinal (GI) tract is primarily dedicated to the processing and reabsorption of nutrients from food, the GI tract also maintains central roles in the integrity of the vascular, lymphatic, and nervous systems and of numerous other organs through its mucosal and submucosal immune system1. The GI immune system has an influential role in both gastrointestinal and systemic health due to its constant exposure to foreign antigens from food, commensal bacteria, or invading pathogens1,2. Thus, the GI immune system must maintain a delicate balance in which it tolerates non-pathogenic antigens while responding appropriately to pathogenic antigens1,2. When the balance of tolerance and defense is disrupted, localized or systemic immune dysregulation and inflammation can occur resulting in a myriad of diseases1,2,3.
The intestine harbors at least 70% of all lymphoid cells in the body4. Most primary immunologic interactions involve at least one of three immune stations in the intestine: 1) Peyer&#8217;s Patches, 2) Intraepithelial lymphocytes (IEL) and 3) lamina propria lymphocytes (LPL). Each of these is comprised of a complex interconnected network of immune cells that rapidly respond to normal immune challenges in the gut5. Restricted to the stroma above the muscularis mucosae, the loosely structured lamina propria is the connective tissue of the gut mucosa and includes scaffolding for the villus, the vasculature, lymphatic drainage, and mucosal nervous system, as well as many innate and adaptive immune subsets6,7,8,9. LPL are comprised of CD4+ and CD8+ T cells in an approximate ratio of 2:1, plasma cells and myeloid lineage cells including, dendritic cells, mast cells, eosinophils and macrophages6.
There is a growing interest in understanding the immune dysregulation and inflammation of the gut as it pertains to various disease states. Such conditions as Crohn&#8217;s disease and ulcerative colitis all manifest varying levels of colonic inflammation10,11,12. Additionally, patients with malignant or non-malignant disorders of the marrow or immune system who undergo an allogeneic bone marrow transplantation (allo-BMT) can develop various forms of colitis including 1) direct toxicity from conditioning regimens before BMT, 2) infections caused by immunosuppression after BMT and 3) graft-versus-host disease (GVHD) driven by donor-type T cells reacting to donor allo-antigens in the tissues after BMT13,14,15. All these post-BMT complications result in significant alterations in the immune milieu of the intestines16,17,18. The proposed method allows a dependable assessment of immune cell accumulation in the mouse colon and, when applied to murine recipients after BMT, facilitates an efficient assay of both donor and recipient immune cells involved in transplant tolerance19,20. Additional causes of gut inflammation include malignancies, food allergies, or disruption of the gut microbiome. This protocol allows access of gut mononuclear cells from the colon and, with modifications, to leukocytes of the small intestine in any of these preclinical murine models.
A PubMed search using the search terms &#8220;intestine AND immune cell AND isolation&#8221; reveals over 200 publications describing methods for small intestine digestion to extract immune cells. However, a similar literature search for colon yields no well-delineated protocols specifying isolation of immune cells from the colon. This may be because the colon has more muscular and interstitial layers, rendering it more difficult to completely digest than the small intestine. Unlike existing protocols, this protocol specifically uses Collagenase E from Clostridium histolyticum without other bacterial collagenases (Collagenase D/ Collagenase I). We demonstrate that, using this protocol, digestion of the colonic tissue can be achieved while preserving the quality of isolated gut mononuclear immune cells (MNC) without the addition of anti-clumping reagents such as sodium versenate (EDTA), Dispase II, and deoxyribonuclease I (DNAse I)21,22,23. This protocol is optimized to allow reproducible robust extraction of viable MNC from the murine colon for further directed studies and should lend itself to the study of immunology of the colon or (with modifications) the small intestine24,25.

<table>
	<tbody>
		<tr>
			<td>60 mm Petri DIsh</td>
			<td>Thermo Scientific</td>
			<td>150288</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Lonza BioWhittaker</td>
			<td>BW17-517Q</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Disposable Serological Pipette</td>
			<td>Corning</td>
			<td>4100</td>
			<td></td>
		</tr>
		<tr>
			<td>10mL Syringe</td>
			<td>Becton Dickinson</td>
			<td>302995</td>
			<td></td>
		</tr>
		<tr>
			<td>15mL Non-Sterile Conical Tubes</td>
			<td>TruLine</td>
			<td>TR2002</td>
			<td></td>
		</tr>
		<tr>
			<td>18- gauge Blunt Needle</td>
			<td>Becton Dickinson</td>
			<td>305180</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL Disposable Serological Pipette</td>
			<td>Corning</td>
			<td>4250</td>
			<td></td>
		</tr>
		<tr>
			<td>40 micrometer pore size Cell Strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon Tube</td>
			<td>Corning</td>
			<td>21008-951</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A4503-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixation Buffer</td>
			<td>Biolegend</td>
			<td>420801</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli Collagenase E from Clostridium histolyticum</td>
			<td>Sigma</td>
			<td>C2139</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 0.5M Sterile Solution</td>
			<td>Amresco</td>
			<td>E177-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo /Fisher Scientific -HyCLone</td>
			<td>SV30014.03</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>GE Healthcare-HyClone</td>
			<td>SH30237.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare-Life Sciences</td>
			<td>1708901</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Medium</td>
			<td>Corning</td>
			<td>17-105-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>VWR Life Science Amresco</td>
			<td>97064-646</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Lonza BioWhittaker</td>
			<td>17-942E</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of lamina propria mononuclear cells from murine colon using collagenase e

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

When working with murine colon disease models, it is helpful to be able to both quantify and qualitatively assess, among the MNC of the colon, multiple immune cell subsets involved in the inflammatory process. The single-cell suspension of MNC obtained through the application of this protocol facilitates such phenotypic characterization in a robust and reproducible manner. As a proof of principle for the application of this isolation method under diverse experimental settings, we retrieve...

Watch this Scientific Journal Video about Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E at JoVE.com

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

isolation-lamina-propria-mononuclear-cells-from-murine-colon-using

Research

JoVE Journal

Immunology and Infection

15.5K Views.  University of Miami Miller School of Medicine. The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

Video: Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...