Name: isolation of mouse kidney-resident cd8+ t cells for flow cytometry analysis
Uploaded: 2020-06-27T14:00:00.000+00:00
Duration: 6 min 7 s
Description: Watch this Scientific Journal Video about Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis at JoVE.com

This work is supported by NIH grants AI125701 and AI139721, Cancer Research Institute CLIP program and American Cancer Society grant RSG-18-222-01-LIB to N.Z. We thank Karla Gorena and Sebastian Montagnino from Flow Cytometry Facility. Data generated in the Flow Cytometry Shared Resource Facility were supported by the University of Texas Health Science Center at San Antonio (UTHSCSA), NIH/NCI grant P30 CA054174-20 (Clinical and Translational Research Center [CTRC] at UTHSCSA), and UL1 TR001120 (Clinical and Translational Science Award).

All animal experiments performed following this protocol must be approved by the respective institutional Animal Care and Use Committee (IACUC). All procedures described here have been approved by IACUC UT Health San Antonio.
1. Adoptive transfer of P14 T cells into C57BL/6 recipients and LCMV infection
<ol>
	<li>Use P14 mice at 6-12 weeks of age. Ensure that the sex of donor P14 TCR transgenic mouse should match the sex of C57BL/6 recipient mice. Otherwise, cells such as male T cells transferring into female mice will result in immune-mediated rejection of circulating donor T cells in around 12 days14.</li>
	<li>Purify na&#239;ve CD8+ T cells from the spleen and lymph nodes of a P14 TCR transgenic mouse using mouse CD8+ T cell purification kit following the manufacturer&#8217;s protocol.
	<ol>
		<li>Briefly, dissect spleen and lymph nodes, mash the samples to generate single cell suspension.</li>
		<li>To enrich for na&#239;ve T cells, add biotin-anti-CD44 antibody during the first antibody incubation step13. 
		NOTE: Negative selection commercial kit used in this step contains two major components for two steps of incubation (i.e., biotin-antibody mixture incubation and streptavidin-magnetic bead incubation).</li>
	</ol>
	</li>
	<li>Use a hemocytometer to count the cells and inject 1,000 to 10,000 purified P14 T cells in 200 &#181;L of PBS into each sex matched C57BL/6 recipient via the tail vein. 
	NOTE: Detailed tail vein injection protocol is described in step 2.</li>
	<li>On the next day, all C57BL/6 recipients receive 2 x 105 pfu LCMV Armstrong diluted in 200 &#181;L of PBS via an intraperitoneal route.</li>
</ol>
2. Intravascular labeling of CD8+ T cells
NOTE: Depending on the research interests, different time points can be chosen following infection. An example of day 30 post infection (day 30 after step 1.4) is demonstrated, which is often considered as an early memory time point for CD8 T cell response.
<ol>
	<li>Dilute biotin anti-CD8&#945; antibody to 15 &#181;g/mL in PBS. Ensure each mouse receive 200 &#956;L of PBS containing 3 &#181;g antibody. 
	NOTE: Biotin anti-CD8 antibody is used here so that it will be more flexible to design the final FACS staining panel. Fluorescent dye-labeled antibody can be used directly. However, it is important to use different clones of monoclonal antibodies for intravenous labeling versus FACS staining.</li>
	<li>Properly heat the tail vein of mice with an overhead heat lamp for 5-10 min to dilate the veins. 
	NOTE: Extra care must be taken to prevent overheating the animals. Reduce the heat or remove the heat lamp if the mice stopped to move around.</li>
	<li>Draw 200 &#181;L of prediluted anti-CD8&#945; antibody mix into a 100 U insulin syringe (28G) and remove any air bubbles by moving the piston up and down. Bend the needle to create a 150&#176; angle between the needle and syringe, bevel up, so the needle will be parallel to the vein.</li>
	<li>Restrain the mouse with a rodent restrainer of appropriate size and spray the tail with 70% ethanol to make the vein clearly visible. 
	NOTE: The duration of the restraint should be kept to a minimum.</li>
	<li>Hold the tail at the distal end with thumb and middle fingers of the non-dominant hand. Place the index finger underneath the site where the needle will be inserted.</li>
	<li>Hold the syringe with the dominant hand and insert the needle into the vein in parallel towards the direction of the heart. 
	NOTE: When placing correctly, the needle should move smoothly into the vein.</li>
	<li>Slowly inject 200 &#181;L of anti-CD8&#945; antibody via the tail vein. 
	NOTE: If there is a resistance or a white area is seen above the needle on the tail, the needle is not inside the vein. Start the initial injection close to the tip of the tail. When necessary, move forward towards the bottom of the tail for subsequent injections.</li>
	<li>Remove the needle and gently apply compression until bleeding stops.</li>
	<li>Return the mouse to the cage, and euthanize the mouse after 3-5 min. 
	NOTE: The mice should be euthanized via isoflurane chamber followed by cervical dislocation. Other methods, such as CO2 inhalation will take up to 5 min and may introduce unnecessary variation of intravenous antibody labeling time.</li>
	<li>Dissect the kidney using scissors, transfer the whole kidney to 1.5 mL of microcentrifuge tubes and leave the samples on ice until further processing. 
	NOTE: Intact whole kidney can be safely stored on ice for a few hours before subsequent processing and digestion.</li>
</ol>
3. Enzymatic digestion of the kidney
<ol>
	<li>Prepare 6 well plates containing 3mL of the digestion solution (RPMI/2% FCS/1 mg/ml Collagenase B) for each mouse, store on ice. 
	NOTE: Stock collagenase B solution at higher concentration (e.g., 20x) can be prepared and stored at or below -20&#176;C. Working solution should be prepared fresh. Please see Table 1 for the recipes for all solution/buffer used in the current protocol.</li>
	<li>Add 300 &#956;L of digestion solution into the 1.5 mL microcentrifuge sample tubes and mince the kidney samples with a straight spring scissor. 
	NOTE: The kidney tissue should be minced into even pieces with sizes no larger than 1.5 mm in diameters. No decapsulation step is required.</li>
	<li>Transfer the minced kidney samples to 6 well plates containing the digestion solution 
	NOTE: 6 well plates are used for convenience only when there are multiple samples to be processed.</li>
	<li>Incubate the samples at 37 &#176;C with gentle rocking (around 60 rpm) for 45 min.</li>
	<li>After digestion, mash the tissue with the plunger flange of a 3 mL syringe directly inside the dish, and transfer into 15 mL conical tubes. 
	NOTE: Usually filtration through a cell strainer is not required at this step because density gradient centrifugation is performed next to purify live lymphocytes.</li>
	<li>Spin down the samples at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Resuspend the pellet in 3 mL of RPMI/10% FCS.</li>
</ol>
4. Density gradient centrifugation to enrich lymphocytes from the digested kidney
<ol>
	<li>Spin down the sample at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Remove the supernatant and resuspend the cell pellet with 5 mL of 44% Percoll/RPMI mix (44% Percoll + 56% RPMI without FBS).</li>
	<li>Put the tip of a 3 mL pipette containing 3 mL of 67% Percoll/PBS (67% Percoll + 33% PBS) directly to the bottom of each tube, and slowly release the solution so that the heavy solution (67% Percoll/PBS) forms a distinct layer at the bottom and the light solution (44% Percoll/RPMI) is raised up. Together, cocktail layers will form. 
	NOTE: For newly arrived density gradient medium from the vendor, add 1/10 volume of sterile 10x PBS to the bottle, mix well and consider the PBS-balanced density gradient medium as 100% solution.</li>
	<li>Spin the samples at 900 x g for 20 min at room temperature with reduced accelerator and brake setting. 
	NOTE: For centrifuges with accelerator and brake settings (on a 1-9 scale (1 means minimum and 9 means maximum)), use 6 for accelerator and 4 for brake.</li>
	<li>Carefully remove the tubes from the centrifuge without disturbing the layers; 
	NOTE: A clear lymphocyte layer is identified between pink color RPMI layer and colorless PBS layer.</li>
	<li>Remove the top layer with a transfer pipette without touching the lymphocyte layer.</li>
	<li>Transfer the lymphocyte layer to a new 15 mL tube, fill the tube with PBS/5% FCS and mix by inverting the tube 4-6x slowly to ensure sufficient mixing. 
	NOTE: This step is important to wash out any residual Percoll.</li>
	<li>Spin down the samples at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Remove the supernatant and re-suspend the cell pellet with 500 &#956;L of complete RPMI medium. The cells are ready for flow cytometry staining.</li>
</ol>
5. Flow cytometry staining and analysis
NOTE: Please follow standard flow cytometry staining protocol for T lymphocytes.
<ol>
	<li>Take around 1 x 106 cells for each FACS staining. 
	NOTE: Use a U-bottom 96 well plate for FACS staining. However, if only a limited number of samples are involved, 5 mL FACS tubes can also be used.</li>
	<li>Spin down the cells at 500 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend each sample in 50 &#181;L of FACS buffer containing 10 &#181;g/mL of 2.4G2 FcR blocker (an anti-CD16/32 monoclonal antibody to block the interaction between added FACS antibody with FcR). Incubate on ice for 15 min.</li>
	<li>Without further centrifugation, add 50 &#181;L of surface staining antibody mixture for each sample so that the final staining volume is 100 &#181;L/each sample. Incubate on ice for 30 min in the dark. 
	NOTE: Include fluorescently labeled streptavidin (2 &#181;g/mL or follow manufacturer&#8217;s recommendation) in the antibody mixture to label CD8&#945;+ intravascular CD8+ T cells and use fluorescent labeled anti-CD8&#946; antibody to label all CD8 T cells. Antibody mixture is made by adding desired amounts of interested antibodies into the FACS buffer.</li>
	<li>Wash the samples with PBS twice. For each wash, fill the wells of 96 well plate with cold PBS, spin at 500 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
	<li>Resuspend the cells in 100 &#181;L/well of diluted live/death dye. Incubate on ice for 30 min in the dark. 
	NOTE: Even with density gradient medium spin, enzymatic digestion step often induces significant cell death in tissue-resident T cells due to ARTC2.2/P2RX7 signaling15. Live/death dye staining is highly recommended before fixation. Anti-ARTC2 nanobody can be administrated into the mice before euthanasia to prevent cell isolation-induced cell death.</li>
	<li>Wash the samples with FACS buffer twice. For each wash, fill the wells of 96-well plate with cold FACS buffer, spin at 500 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
	<li>Resuspend the cells in 100 &#956;L/well fixing buffer. Incubate at 37 &#176;C for 10 min. 
	NOTE: Fixing buffer is made freshly by diluting formaldehyde with PBS to a final concentration of 2% formaldehyde.</li>
	<li>Wash the samples with FACS buffer twice. For each wash, fill the wells of 96 well plate with cold FACS buffer, spin at 500 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
	<li>Resuspend the cells in 250 &#956;L/well FACS buffer. Seal the plate, store at 4 &#176;C and protect from light until FACS analysis. 
	NOTE: Before FACS analysis, filter the samples through a 70 &#956;M nylon mesh to remove possible cell clusters that may clog the FACS machine.</li>
</ol>

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The authors have no relevant financial disclosures.

As tissue specific immunity is a rapid expanding area of research, accumulating evidence suggest that immune cells, especially lymphocytes population can be identified in almost all organs in adult human or infected or immunized mice. LCMV mouse infection model is a well-established model to study antigen-specific T cell response, effector and memory T cell differentiation including TRM biology across multiple tissues. Here, we described a protocol to analyze CD8+ T cells in the kidney. This protoco...

Tissue-resident memory (TRM) T cells represent one of the most abundant T cell populations in adult human and infected mice. TRM cells provide the first line of immune defense and are critically involved in various physiological and pathological processes1,2,3,4,5. Comparing with circulating T cells, TRM cells carry distinct surface markers with unique transcription programs6,7,8. Expanding our knowledge of TRM biology is the key to understanding T cell responses which is essential for future development of T cell-based vaccines and immunotherapies.
In addition to commonly shared molecular markers of TRMs across all non-lymphoid tissues, accumulating evidence suggest that tissue-specific features are a central component of TRM biology9. Kidney harbors many immune cells including TRM cells after infection and offers a great opportunity to study TRM cell biology in a non-mucosal tissue. Acute LCMV (lymphocytic choriomeningitis virus) infection via intraperitoneal route is a well-established systemic infection model to study antigen-specific T cell responses in mice. The infection is usually resolved in 7-10 days in wild type mice and leave a large number of LCMV-specific memory T cells in a variety of tissues, including the kidney10. P14 TCR (T cell receptor) transgenic mice carry CD8+ T cells recognize one of the immune-dominant epitopes of LCMV presented by MHC-I (class I major histocompatibility complex) molecule H2-Db in C57BL/6 mice. Combining congenically marked P14 T cell adoptive transfer and LCMV infection in mice, CD8+ effector and memory T cells are tracked, including TRM differentiation and homeostasis.
In some barrier tissues, such as intestinal intraepithelial lymphocytes (IEL) compartment and salivary glands, established lymphocyte isolation procedure yields high percentage of TRM cells with minimal blood borne T cell contamination in mouse LCMV model11. However, in non-barrier tissues, such as the kidney, dense vascular network contains a large number of circulating CD8+ T cells. It has been well documented that even successful perfusion cannot efficiently remove all circulating CD8+ T cells. To overcome this technical hurdle, intravascular antibody staining has been established as one of the most commonly used techniques in TRM labs12. In brief, 3-5 minutes before euthanasia, 3 &#181;g/mouse anti-CD8 antibody (to label CD8+ T cells) is delivered intravenously. Intact blood vessel wall prevents rapid diffusion of the antibody within this short period of time (i.e., 3-5 minutes) and only intravascular cells are labeled. Following standard lymphocyte isolation protocol, intravascular versus extravascular cells can be easily distinguished using flow cytometry.
Here, we will describe a standard protocol commonly used in TRM labs to perform intravascular labeling, lymphocyte isolation and flow cytometry analysis of kidney CD8+ T cells using a C57BL/6 mouse that has received CD45.1+ P14 T cells and LCMV infection 30 days before13. Same protocol can be used to study both effector and memory T cells in the kidney.

<table><tbody><tr><td>1.5 ml microcentrifuge tubes</td><td>Fisherbrand</td><td>05-408-130</td><td></td></tr><tr><td>15 ml Conical Tubes</td><td>Corning</td><td>431470</td><td></td></tr><tr><td>3 ml syringe</td><td>BD</td><td>309657</td><td></td></tr><tr><td>37C incubator</td><td>VWR</td><td></td><td></td></tr><tr><td>Biotin a-CD8a antibody(Clone 53-6.7)</td><td>Tonbo Biosciences</td><td>30-0081-U025</td><td></td></tr><tr><td>Calf Serum</td><td>GE Healthcare Life Sciences</td><td>SH30073.03</td><td></td></tr><tr><td>Collegenase B</td><td>Millipore Sigma</td><td>11088807001</td><td></td></tr><tr><td>Disposable Graduated Transfer Pipettes</td><td>Fisherbrand</td><td>13-711-9AM</td><td></td></tr><tr><td>Insulin Syringe</td><td>BD</td><td>329424</td><td></td></tr><tr><td>Micro Dissecting Spring Scissors</td><td>Roboz Surgical</td><td>RS 5692</td><td></td></tr><tr><td>Mojosort Mouse CD8 Na&amp;iuml;ve T Cells Isolation Kit</td><td>Biolegend</td><td>480043</td><td></td></tr><tr><td>overhead heat lamp</td><td>Amazon</td><td>B00333PZZG</td><td></td></tr><tr><td>PBS</td><td></td><td></td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare Life Sciences</td><td>17089101</td><td></td></tr><tr><td>rocker</td><td>VWR</td><td></td><td></td></tr><tr><td>RPMI</td><td>GE Healthcare Life Sciences</td><td>SH30096.01</td><td></td></tr><tr><td>Solid Brass Mouse Restrainer</td><td>Braintree Scientific, Inc</td><td>SAI-MBR</td><td></td></tr><tr><td>Swing Bucket Centrifuge with refrigerator</td><td>Thermofisher</td><td></td><td></td></tr><tr><td>Tissue Culture 6-well plate</td><td>Corning</td><td>3516</td><td></td></tr></tbody></table>

isolation of mouse kidney-resident cd8+ t cells for flow cytometry analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

The protocol described here is summarized in a flow chart (Figure 1A). At day 30 post LCMV infection, we performed intravascular labeling of CD8+ T cells. 5 minutes later, both kidneys of the animal were dissected, minced and subjected to collagenase digestion. Lymphocytes were further purified from the digested samples via Percoll centrifugation and analyzed by flow cytometry. As shown in Figure 1B, even after density centrifugation-mediated lymphocy...

Watch this Scientific Journal Video about Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis at JoVE.com

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

isolation-mouse-kidney-resident-cd8-t-cells-for-flow-cytometry

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Immunology and Infection

6.6K Views.  University of Texas Health Science Center at San Antonio. Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

Video: Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of acute leukemia. However, the nature of this disease has not been well documented in the literature. Here, we provide a technical protocol for characterizing C1498 cells in vitro and for determining the nature of the induced leukemia in vivo. The first part of this procedure is focused on determining the hematopoietic lineage and the stage of differentiation of cultured C1498 cells. To achieve this, multi-parametric flow cytometric staining is used to detect hematopoietic cell markers. Immunofluorescence microscopy, cytochemistry and a May-Gr&#252;nwald Giemsa staining are then performed to assess the expression of myeloperoxidase, the activity of esterases and cellular morphology, respectively. The second part of this protocol is dedicated to describing the leukemia disease that is induced in vivo. The latter can be achieved by determining the frequencies of leukemic and inherent cells in the blood, hematopoietic organs (e.g., bone marrow and spleen) and non-lymphoid tissues (e.g., the liver and lungs) using specific staining and flow cytometry analyses. The nature of the leukemia is then confirmed using May-Gr&#252;nwald Giemsa staining and staining for specific esterases in the bone marrow. Here, we present the results that were obtained using this protocol in age-matched C1498- and PBS-injected mice.

This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Gr&#252;nwald Giemsa staining.

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

Cancer Research

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Reliable and High Efficiency Extraction of Kidney Immune Cells

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...

Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to assess calcium signaling by band-pass flow cytometry. This protocol is designed to measure calcium responses in primary murine T-cells using full spectrum flow cytometry. Total splenocytes are labeled with the ratiometric calcium indicator dye Indo-1, along with a panel of fluorochrome-conjugated antibodies to cell surface molecules. Leveraging the capabilities of full spectrum flow cytometry provides a platform for utilizing a wide array of cell surface stains in combination with Indo-1. Cells are then analyzed in real-time at 37 &#176;C before and after the addition of an anti-CD3 antibody to stimulate the T-cell receptor. After unmixing the spectral signals, the ratio of calcium-bound to calcium-free Indo-1 is calculated and can be visualized over time for each gated population of splenocytes. This technique can allow for the simultaneous analysis of calcium responses in multiple cell populations.

Calcium influx, a measure of T-cell signaling, is an effective way to analyze responses to T-cell receptor stimulation. This protocol for multiplexing Indo-1 with panels of antibodies directed at cell surface molecules takes advantage of the highly flexible capabilities of full spectrum flow cytometry.

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to ...

Elucidating T-Cell Kinetics: Methods for Real-Time Migration Analysis

Real-Time In Vitro Migration Assay for Primary Murine CD8+ T Cells

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign bodies. T cell migration is a complex process that requires the coordination of many signal inputs from the environment and local immune cells, including chemokines, chemokine receptors, and adhesion molecules. Furthermore, T cell motility is influenced by dynamic surrounding environmental cues, which can alter activation state, transcriptional landscape, adhesion molecule expression, and more. In vivo, the complexity of these seemingly intertwined factors makes it difficult to distinguish individual signals that contribute to T cell migration. This protocol provides a string of methods from T cell isolation to computer-aided analysis to assess T cell migration in real-time&#160;under highly specific environmental conditions.&#160;These conditions&#160;may help&#160;elucidate mechanisms that regulate migration, improving our&#160;understanding of T cell kinetics and providing&#160;strong mechanistic evidence that is difficult&#160;to attain through animal experiments. A deeper understanding of the&#160;molecular interactions that impact cell&#160;migration is important to develop improved therapeutics.

Author Spotlight: Understanding Disease Mechanisms Through Real-Time Analysis of T-Cell Migration

This protocol providesa method of primary murine T cell isolation and time-lapse microscopy of T cell migration under specific environmental conditions with quantitative analysis.

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign ...

Isolation of Immune and Non&mdash;Immune Cells from Mouse Lungs for Flow Cytometry

Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development of disease. Studies utilizing mouse models of respiratory infection, allergy, fibrosis, and cancer have been critical to reveal mechanisms of disease progression and identify therapeutic targets. However, most studies focused on the mouse lung prioritize the isolation of either immune cells or epithelial cells, rather than both populations concurrently. Here, we describe a method for preparing a comprehensive single-cell suspension of both immune and non-immune populations suitable for flow cytometry and fluorescence-activated cell sorting. These populations include epithelial cells, endothelial cells, fibroblasts, and a variety of myeloid cell subsets. This protocol entails bronchoalveolar lavage and subsequent inflation of the lungs with dispase. Lungs are then digested in a liberase mixture. This method of processing liberates a variety of diverse cell types and results in a single-cell suspension that does not require manual dissociation against a filter, promoting cell survival and yielding high numbers of live cells for downstream analyses. In this protocol, we also define gating schemes for epithelial and myeloid cell subsets in both na&#239;ve and influenza-infected lungs. Simultaneous isolation of live immune and non-immune cells is key for interrogating intercellular crosstalk and gaining a deeper understanding of lung biology in health and disease.

This protocol details the isolation of live immune and non-immune populations from the mouse lung at a steady state and following influenza infection. It also provides gating strategies for identifying epithelial and myeloid cell subsets.

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development ...

Isolation of Mouse Kidney-Resident CD8⁺ T cells for Flow Cytometry Analysis

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