Name: isolation of mouse kidney-resident cd8+ t cells for flow cytometry analysis
Uploaded: 2020-06-27T14:00:00
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 transf...

<ol>
	<li>Mueller, S. N., Gebhardt, T., Carbone, F. R., Heath, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+T+cell+subsets,+migration+patterns,+and+tissue+residence.">Memory T cell subsets, migration patterns, and tissue residence.</a> Annual Review of Immunology. 31, 137-161 (2013).</li><li>Mueller, S. N., Mackay, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+memory+T+cells:+local+specialists+in+immune+defence.">Tissue-resident memory T cells: local specialists in immune defence.</a> Nature Reviews: Immunology. 16, 79-89 (2016).</li><li>Park, C. O., Kupper, T. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Runx3+programs+CD8(+)+T+cell+residency+in+non-lymphoid+tissues+and+tumours.">Runx3 programs CD8(+) T cell residency in non-lymphoid tissues and tumours.</a> Nature. 552, 253-257 (2017).</li><li>Liu, Y., Ma, C., Zhang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-Specific+Control+of+Tissue-Resident+Memory+T+Cells.">Tissue-Specific Control of Tissue-Resident Memory T Cells.</a> Critical Reviews in Immunology. 38, 79-103 (2018).</li><li>Steinert, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Memory+CD8+T+Cells+Reveals+Regionalization+of+Immunosurveillance.">Quantifying Memory CD8 T Cells Reveals Regionalization of Immunosurveillance.</a> Cell. 161, 737-749 (2015).</li><li>Qiu, Z., Sheridan, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating+Lymphocytes+from+the+Mouse+Small+Intestinal+Immune+System.">Isolating Lymphocytes from the Mouse Small Intestinal Immune System.</a> Journal of Visualized Experiments. (132), e57281 (2018).</li><li>Anderson, K. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+staining+for+discrimination+of+vascular+and+tissue+leukocytes.">Intravascular staining for discrimination of vascular and tissue leukocytes.</a> Nature Protocols. 9, 209-222 (2014).</li><li>Ma, C., Mishra, S., Demel, E. L., Liu, Y., Zhang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+Controls+the+Formation+of+Kidney-Resident+T+Cells+via+Promoting+Effector+T+Cell+Extravasation.">TGF-beta Controls the Formation of Kidney-Resident T Cells via Promoting Effector T Cell Extravasation.</a> Journal of Immunology. 198, 749-756 (2017).</li><li>Gebhardt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+patterns+of+peripheral+migration+by+memory+CD4++and+CD8++T+cells.">Different patterns of peripheral migration by memory CD4+ and CD8+ T cells.</a> Nature. 477, 216-219 (2011).</li><li>Borges da Silva, H., Wang, H., Qian, L. J., Hogquist, K. A., Jameson, S. 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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalizing+the+environment+recapitulates+adult+human+immune+traits+in+laboratory+mice.">Normalizing the environment recapitulates adult human immune traits in laboratory mice.</a> Nature. 532, 512-516 (2016).</li></ol>

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&#239;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 ...

CD8+T cells carry distinct features in different tissue, as a critical example of non-mucosal and non-lymphoid tissue. It is essential to directly characterize kidney-resident cells to fully understand T-cell biology. Here we will demonstrate our convenient method to isolate kidney-resident CD8+T cells accommodating subsequent flow cytometry analysis.This method could provide insight into the studies of effector and memory CD8+T cells generated after infections as well as autoimmune responses. The procedure will be performed by Dr.Chaoyu Ma, a research scientist for my laboratory. Begin by diluting biotin anti CD8 alpha antibody to 15 micrograms per milliliter in PBS, making sure that there is enough to administer 200 microliters to each mouse.Prior to injection, heat the tail vein of the mouse with an overhead heat lamp for five to 10 minutes to dilate it. Draw 200 microliters of the pre diluted antibody mix into a 28-gauge insulin syringe and remove any air bubbles by moving the piston up and down. Bend the needle to create a 150-degree angle between the needle and the syringe, bevel up, so that the needle is parallel to the tail vein.Restrain the mouse with a rodent restrainer and spray the tail with 70%ethanol to make the vein clearly visible. Hold the tail at the distal end with the thumb and middle fingers of the non-dominant hand, then place the index finger underneath the injection site. With the dominant hand insert the needle into the vein in parallel towards the direction of the heart and slowly inject 200 microliters of the antibody.Remove the needle and gently compress the injection site until bleeding stops. After euthanizing the mouse, dissect the kidney with scissors and transfer it to a 1.5-milliliter micro centrifuge tube. Leave the samples on ice until further processing.Prepare six-well plates with three milliliters of digestion solution in each well, using one well per mouse, and store them on ice. Add 300 microliters of digestion solution into each sample tube and mince the kidney samples with a straight spring scissor. Transfer the minced kidney samples to the six-well plates with the digestion solution.Incubate the samples at 37 degrees Celsius with gentle rocking for 45 minutes, then homogenize the tissue with the plunger flange of a three-milliliter syringe and transfer it to 15-milliliter conical tubes. Spin the samples down at 500 times g and four degrees Celsius for five minutes. Remove the supernatant and resuspend the pellet in three milliliters of RPMI with 10%FCS.Spin down the sample at 500 times g and four degrees Celsius for five minutes, then remove the supernatant and resuspend the cell pellet with five milliliters of 44%density gradient medium and RPMI mix. Put the tip of a three-milliliter pipette containing three milliliters of 67%density gradient medium and PBS directly to the bottom of each tube and slowly release the solution so that the heavy solution forms a distinct layer at the bottom. Spin the samples at 900 times g for 20 minutes with reduced accelerator and brake settings, then carefully remove the tubes from the centrifuge without disturbing the layers.Remove the top layer with a transfer pipette without touching the lymphocyte layer and transfer the lymphocyte layer to a new 15-milliliter tube. Fill the tube with PBS and 5%FCS, then mix by slowly inverting the tube four to six times. After centrifuging the samples at 500 times G and four degrees Celsius for five minutes, remove the supernatant and resuspend the cell pellet with 500 microliters of complete RPMI medium.The cells are now ready for flow cytometry staining. Even after density centrifugation-mediated lymphocyte enrichment, it was very common to see a large portion of non lymphocytes in the final product. However, after gaining on live lymphocytes, CD8+T lymphocytes were easy to identify.As expected, only extra-vascular CD8+T cells efficiently acquired tissue resident memory T-cell phenotypes, such as upregulation of CD69 and downregulation of Ly6C. In contrast to the infected mice, the vast majority of CD8+T cells isolated from young naive mice housed in the SPF facility were labeled with CD8 alpha antibody and therefore belonged to the intravascular compartment. This technique has greatly accelerated the investigation of tissue-resident immune cells in densely vascularized organs.

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Research

JoVE Journal

Immunology and Infection

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

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 ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G0. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G1 that can remain in this phase, proceed into cycling, or move into G0.
Here, we describe a flow cytometric method for capturing a &#34;snapshot&#34; of T cells in different cell cycle phases in mouse secondary lymphoid organs. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G0, in G1 and in S-G2/M phases of the cell cycle in the spleen&#160;and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1.
Critical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen-specific T cells that would have been missed by current criteria of analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discrimination&#160;of the G0/G1 and G2/M DNA peaks, while preserving membrane and intracellular staining. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis.

Clonal expansion is a key feature of antigen-specific T cell response. However, the cell cycle of antigen-responding T cells has been poorly investigated, partly because of technical limitations. We describe a flow cytometric method to analyze clonally expanding antigen-specific CD8 T cells in spleen and lymph nodes of vaccinated mice.

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...