The authors would like to thank Bing Zhang for his technical assistance. This work was funded by the Canadian Institutes for Health Research (MOP-86595). T.A.B is a CIHR New Investigator and AHFMR Scholar. Q.H. is supported by a CIHR Canada Graduate Scholarship - Doctoral and an AIHS Full-time Studentship. S.A.N. is supported by a Queen Elizabeth II Graduate Scholarship. A.Y.W.S. is supported by an NSERC Postgraduate Scholarship - Doctoral.

Refer to Figure 1 for an overall scheme of the experimental protocol.
1. Dissection
<ol>
 <li>Place sterile steel mesh screen into 60 x 15 mm Petri dish. One unit is needed per tissue sample.</li>
 <li>Add 5 ml of Hank's balanced salt solution (HBSS) to each dish. Keep dishes on ice.</li>
 <li>Euthanize mice with CO2.</li>
 <li>Secure mouse to dissection surface, ventral side facing up. Spray mouse with 70% ethanol for sterilization and to ensure that the fur is matted down.</li>
 <li>Using surgical scissors, begin dissection by making a ventral incision in the abdominal skin just above the genitalia. Extend the incision upwards to the chin. </li>
 <li>From the midline, extend the incision down along all limbs. Pull back the loose skin and pin it down to the dissection surface.</li>
 <li>Harvest the thymus:
 <ol>
 <li>Lift the bottom tip of the sternum to make an incision.</li>
 <li>Avoiding the liver, cut the diaphragm to detach the rib cage and then cut the rib cage to each side. Cut the ribcage upwards on each side, taking care to avoid the lungs and heart.</li>
 <li>Gently pull the ribcage back with forceps. The thymus is a white, bilobed organ located above the heart. Using the flat edge of the forceps to grasp the bottom of the lobes, gently pull the thymus and place it on the mesh screen.</li>
 </ol>
 </li>
 <li>Harvest the spleen:
 <ol>
 <li>The spleen is a red, surfboard-shaped organ located on the left side of the mouse's abdominal cavity, below the liver.</li>
 <li>Make an incision into the abdominal cavity. Gently pull out the spleen with one pair of forceps, using the second pair to tease away connective tissue. Place the spleen on a separate mesh screen.</li>
 </ol>
 </li>
</ol>
2. Cell Preparation
<ol>
 <li>Using a plunger from a 3 ml syringe, grind organs into the mesh screen until only connective tissue and fat remains. Rinse mesh with HBSS from the dish three times. Use a new plunger for each tissue sample.
 <ol>
 <li>Other options for homogenizing tissue can be used in place of this method.</li>
 </ol>
 </li>
 <li>Count cells using a hemocytometer.</li>
 <li>Pellet cells by centrifugation at 335 x g for 5 min at 4 &deg;C.</li>
 <li>Resuspend splenocytes in 500 &mu;l of ACK lysis buffer (0.15 M NH4Cl, 10 mM KHC03, 0.1 mM Na2EDTA, pH 7.2) for 10 min at room temperature. Resuspend thymocytes at 20 x 106 cells/ml in FACS buffer (PBS, 1% FCS, 0.02% sodium azide) and set aside on ice.</li>
 <li>Return splenocytes to isotonicity by adding 5 ml of HBSS. Pellet splenocytes by centrifugation and resuspend at 20 x 106 cells/ml in FACS buffer. If cells need to remain sterile, you may use sterile RPMI + 10% FCS.</li>
</ol>
3. Staining Cells for Flow Cytometry
The purpose of this protocol is to outline strategies for the analysis of positive and negative selection using wildtype (WT) and HYcd4 mice. For general considerations regarding flow cytometry experimental design, acquisition, and analysis, we refer readers to an excellent review by Tung et al.11
<ol>
 <li>Aliquot 4 x 106 thymocytes per flow cytometry sample to each well of a 96-well plate, as well as 1 x 106 WT splenocytes per compensation control. If using transgenic mice, you should include a WT mouse as a control in your experiment.</li>
 <li>Block Fc receptors by incubating cells with anti-CD16/32 (clone 2.4G2) for 10 min on ice.</li>
 <li>Spin plate at 335 x g at 4 &deg;C for 5 min. Remove lid and dispel liquid from wells by flicking the plate once, face down into a sink. Resuspend each well in 200 &mu;l of FACS buffer. Repeat the wash.</li>
 <li>Prepare antibody cocktails as listed below, using the optimal concentration of each antibody based on dilution in 200 &mu;l of FACS buffer per well. The optimal concentration of each antibody is defined as the lowest concentration needed to give the largest separation of positive and negative populations. If there is no negative population for a given antibody, an isotype antibody should be included. The total volume per well can be scaled down (e.g. to 100 &mu;l per well) if desired.
 <ol>
 <li>WT thymus: anti-TCR&beta;, anti-CD4, anti-CD8, anti-CD69 or anti-CD5, anti-CD24</li>
 <li>HYcd4 thymus: anti-HY TCR (T3.70), anti-CD4, anti-CD8, anti-CD69 or anti- CD5, anti-CD24 
To obtain better separation of CD69lo and CD69hi populations in thymocytes, it is recommended that a biotinylated anti-CD69 primary antibody be used, followed by a secondary stain containing fluorochrome-conjugated streptavidin. We use the same strategy for CD5 staining.
 </li>
 </ol>
 </li>
 <li>Incubate cells with 200 &mu;l of antibody cocktail in FACS buffer for 30 min on ice in the dark.</li>
 <li>Concurrent with antibody cocktail staining, stain each compensation control with a single fluorochrome-conjugated antibody. Ideally, compensation staining involves the same antibodies as used in the antibody cocktail. However, staining for each individual antigen may not be feasible for larger experiments when many antigens are being assayed. Therefore, staining for either anti-CD4 or anti-CD8 is recommended due to high expression of these antigens, or the use of compensation beads. Stain in FACS buffer for 30 min on ice in the dark. Leave one compensation control unstained. </li>
 <li>Wash cells twice with FACS buffer.</li>
 <li>Resuspend cells in FACS buffer and transfer to FACS tubes.</li>
 <li>Acquire samples on flow cytometer. Include acquisition of FSC-A and FSC-W to allow for doublet discrimination.</li>
</ol>
4. Analysis of Flow Cytometry Data - Non-TCR Transgenic Mice
We use FlowJo for flow cytometry data analysis. Refer to Figure 2A for the gating strategy for non-TCR transgenic mice.
<ol>
 <li>Using FSC-A by SSC, electronically gate on the "lymphocyte" population.</li>
 <li>Within the "lymphocyte" population, use FSC-A by FSC-W to electronically gate the FSC-Wlo population to exclude cell doublets and aggregrates. This is the "singlet" gate.</li>
 <li>Using the events in the "singlet" gate, plot CD8 by CD4. Draw gates for DN, DPdull, DPbright, CD4+CD8lo, CD4SP and CD8SP populations as depicted in Figure 2B.</li>
 <li>Under the CD4SP and CD8SP gates, create TCR&beta; by CD24 plots. Use the quadrant gating tool to draw gates as depicted in Figure 2C.</li>
 <li>Create a new plot from the "singlet" gate: TCR&beta; by CD69. Draw gates A, B, C, D as depicted in Figure 2D.</li>
 <li>Create another plot from the "singlet" gate: TCR&beta; by CD5. Draw gates i, ii, iii, iv as depicted in Figure 2E. This is another strategy for examining positive selection.</li>
 <li>Apply DN, DPdull, DPbright, CD4int, CD4SP and CD8SP gates from under "singlet" to gates i, ii, iii, iv, A, B, C, D (Figure 2D, E).</li>
 <li>Calculate the number of cells in a particular subset by multiplying organ cellularity by the frequency of each subsequent gate until you reach your target population. For example, the number of TCR&beta;hiCD69- CD8SP thymocytes would be determined by thymus cellularity x %TCRbhi CD69- (fraction D) x %CD8SP.
 <ol>
 <li>Counting already takes into account the live and dead cells so do not include the "lymphocyte" gate in your calculations.</li>
 </ol>
 </li>
</ol>
5. Analysis of Flow Cytometry Data - TCR Transgenic Mice
Refer to Figure 3A for the gating strategy for TCR transgenic mice. 
<ol>
 <li>Follow steps 4.1 and 4.2 as in the previous section.</li>
 <li>Under the "singlet" gate, create a T3.70 by SSC plot and gate on the T3.70+ cell population to analyze antigen-specific T cells (Figure 3B). In some circumstances, it may be of interest to compare this population to the non-antigen specific (T3.70-) T cell population.</li>
 <li>Within the T3.70+ population, draw DN, DP, CD4SP and CD8SP gates (Figure 3C).
 <ol>
 <li>For WT control samples, draw these gates under the &#x201C;singlet&#x201D; gate or create a T3.70- gate as there will be very few T3.70+ cells.</li>
 </ol>
 </li>
 <li> Under the CD8SP gate, create a T3.70 by CD24 plot. Use the quadrant gating tool to divide cells into four populations (Figure 3F).</li>
 <li>Create an overlaid histogram depicting CD69 expression in the DP compartment of WT mice and T3.70+ DP compartment of HYcd4 mice (Figure 4A). Repeat to examine CD5 expression (Figure 4B).</li>
 <li>Calculate the absolute number of cells in each subset of interest as in 4.8.</li>
</ol>

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No conflicts of interest declared.

The protocol presented here can be used to examine positive and negative selection in non-TCR transgenic and TCR transgenic mice. This protocol describes the staining of surface antigens. For further analysis of molecular mechanisms, it is often necessary to perform intracellular staining. We use the BD Biosciences Cytofix/Cytoperm Kit for most intracellular proteins and the BD Biosciences Foxp3 Staining Kit for transcription factors. We usually acquire our samples immediately after staining. However, samples can ...

<table><tbody><tr><td>HyClone Hank&#039;s balanced salt solution</td><td>Thermo Scientific</td><td>SH30030.02</td><td></td></tr><tr><td>Metal mesh screens</td><td>Cedarlane</td><td>CX-0080-E-01</td><td></td></tr><tr><td>Petri dishes (60 x 15 mm)</td><td>Fisher Scientific</td><td>877221</td><td></td></tr><tr><td>Syringes (3 ml)</td><td>BD Biosciences</td><td>309657</td><td></td></tr><tr><td>Conical tubes (15 ml)</td><td>Sarstedt</td><td>62.554.205</td><td></td></tr><tr><td>Microscope</td><td>Zeiss - Primo Star</td><td>415500-00XX-000</td><td></td></tr><tr><td>Hemocytometer </td><td>Hausser Scientific</td><td>3110</td><td></td></tr><tr><td>96-well plate</td><td>Sarstedt</td><td>82.1582.001</td><td></td></tr><tr><td>Multichannel pipette</td><td>Fisherbrand</td><td>21-377-829</td><td></td></tr><tr><td>Fetal calf serum</td><td>PAA</td><td>A15-701</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Fisher Scientific</td><td>SH3025802</td><td></td></tr><tr><td>Sodium azide</td><td>IT Baker Chemical Co.</td><td>V015-05</td><td></td></tr><tr><td>FcR blocking reagent</td><td></td><td></td><td>Clone 2.4G2</td></tr><tr><td>Anti-mouse HY TCR </td><td>eBioscience</td><td>XX-9930-YY*</td><td>Clone T3.70</td></tr><tr><td>Anti-mouse CD4</td><td>eBioscience</td><td>XX-0042-YY*</td><td>Clone RM4-5</td></tr><tr><td>Anti-mouse CD8&amp;alpha; </td><td>eBioscience</td><td>XX-0081-YY*</td><td>Clone 53-6.7</td></tr><tr><td>Anti-mouse CD24</td><td>eBioscience</td><td>XX-0242-YY*</td><td>Clone M1/69</td></tr><tr><td>Anti-mouse TCR&amp;#946; </td><td>eBioscience</td><td>XX-5961-YY*</td><td>Clone H57-597</td></tr><tr><td>Anti-mouse CD69 Biotinylated</td><td>eBioscience</td><td>13-0691-YY*</td><td>Clone H1.2F3</td></tr><tr><td>Anti-mouse CD5 Biotinylated</td><td>eBioscience</td><td>13-0051-YY*</td><td>Clone 53-7.3</td></tr><tr><td>Streptavidin</td><td>eBioscience</td><td>XX-4217-YY*</td><td></td></tr><tr><td>Flow cytometer</td><td>BD Biosciences - FACS Canto</td><td>338962</td><td></td></tr><tr><td>FACS tubes</td><td>BD Biosciences</td><td>352052</td><td></td></tr><tr><td>Flow cytometry analysis software</td><td>TreeStar - Flowjo</td><td>FlowJo v7/9</td><td></td></tr><tr><td>HyClone RPMI - 1640 medium</td><td>Thermo Scientific</td><td>SH30027.01</td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;*XX varies by fluorochrome and YY varies by vial size.&lt;/p&gt;</td></tr></tbody></table>

examination of thymic positive and negative selection by flow cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

In physiological TCR transgenic models and WT mice, positive selection begins at the DPbright stage before moving into the DPdull stage after antigen encounter. DPdull thymocytes then enter a transitional CD4+CD8lo stage before becoming CD4SP or CD8SP thymocytes (Figure 2B). Mature SP thymocytes are characterized by high TCR expression and loss of CD24 (Figure 2C). While the CD8 by CD4 profile can reveal defects in positive se...

Watch this Scientific Journal Video about Examination of Thymic Positive and Negative Selection by Flow Cytometry at JoVE.com

Examination of Thymic Positive and Negative Selection by Flow Cytometry

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

examination-thymic-positive-negative-selection-flow

Research

JoVE Journal

Immunology and Infection

21.7K Views.  University of Alberta. We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

Video: Examination of Thymic Positive and Negative Selection by Flow Cytometry

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 ...

Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4). As retroviral transduction requires dividing cells for transgene integration, OP9-DL4 provides a suitable in vitro environment for cultivating hematopoietic progenitor cells. This is particularly advantageous when studying the effects of the expression of a specific gene during normal T cell development and leukemogenesis, as it allows researchers to circumvent the time-consuming process of generating transgenic mice. To achieve successful outcomes, a series of coordinated steps involving the simultaneous manipulation of different types of cells must be carefully performed. Although these are very well-established procedures, the lack of a common source in the literature often means a series of optimizations are required, which can be time-consuming. This protocol has been shown to be efficient in transducing primary thymocytes followed by differentiation on OP9-DL4 cells. Detailed here is a protocol that can serve as a quick and optimized guide for the co-culture of retrovirally transduced thymocytes on OP9-DL4 stromal cells.

This protocol describes the isolation of double-negative thymocytes from the mouse thymus followed by retroviral transduction and co-culture on the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4) for further functional analysis.

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line ...

Cancer Research

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Preparing Mouse Thymic Epithelial Cells for Analysis of Intracellular Molecules

Preparation of Single-Cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric Analysis

Thymic epithelial cells (TECs) play an essential role in promoting the development and repertoire selection of T cells. Cortical TECs (cTECs) in the thymic cortex induce early T cell development and positive selection of cortical thymocytes. In contrast, medullary TECs (mTECs) in the thymic medulla attract positively selected thymocytes from the cortex and establish self-tolerance in T cells. A variety of molecules, including DLL4 and beta5t expressed in cTECs, as well as Aire and CCL21 expressed in mTECs, contribute to thymus function supporting T cell development and selection. Flow cytometric analysis of functionally relevant molecules in cTECs and mTECs is useful to improve our understanding of the biology of TECs, even though current methods for the preparation of single-cell suspensions of TECs can retrieve only a small fraction of TECs (approximately 1% for cTECs and approximately 10% for mTECs) from young adult mouse thymus. Because many of these functionally relevant molecules in TECs are localized within the cells, we describe our protocols for the preparation of single-cell suspension of mouse TECs and the staining of intracellular molecules for flow cytometric analysis.

Preparation of Single-Cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric AnalysisMechanisms

We describe protocols for the preparation of single-cell suspension of mouse thymic epithelial cells and the staining of intracellular molecules for flow cytometric analysis.

Thymic epithelial cells (TECs) play an essential role in promoting the development and repertoire selection of T cells. Cortical TECs (cTECs) in the ...

Quality-Controlled Sputum Analysis by Flow Cytometry

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed using cytology-based methodologies. Its utility is limited because reading the slides is time-consuming and requires highly specialized personnel. Moreover, extensive debris and the presence of too many squamous epithelial cells (SECs), or cheek cells, often renders a sample inadequate for diagnosis. In contrast, flow cytometry allows for high-throughput phenotyping of cellular populations while simultaneously excluding debris and SECs.
The protocol presented here describes an efficient method to dissociate sputum into a single cell suspension, antibody stain and fix cellular populations, and acquire samples on a flow cytometric platform. A gating strategy that describes the exclusion of debris, dead cells (including SECs) and cell doublets is presented here. Further, this work also explains how to analyze viable, single sputum cells based on a cluster of differentiation (CD)45 positive and negative populations to characterize hematopoietic and epithelial lineage subsets. A quality control measure is also provided by identifying lung-specific macrophages as evidence that a sample is derived from the lung and is not saliva. Finally, it has been demonstrated that this method can be applied to different cytometric platforms by providing sputum profiles from the same patient analyzed on three flow cytometers; Navios EX, LSR II, and Lyric. Furthermore, this protocol can be modified to include additional cellular markers of interest. A method to analyze an entire sputum sample on a flow cytometric platform is presented here that makes sputum amenable for developing high-throughput diagnostics of lung disease.

This protocol describes an efficient method for dissociating sputum into a single cell suspension and the subsequent characterization of cellular subsets on standard flow cytometric platforms.

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed ...

Medicine

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the lymph node microenvironment in B-cell non-Hodgkin&#39;s lymphoma is analyzed. Subsets of circulating T-follicular helper cells (cTfh), the circulating memory compartment of Tfh cells in the blood, are also perturbed in disease and therefore represent potential novel predictive biomarkers. Peripheral blood-based testing is advantageous because it is relatively non-invasive and allows simple serial monitoring.This article describes a method for isolating CD4+ T-cells from human blood, and further analysis by flow-cytometry to enumerate cTfh cells and the proportions of their various subsets (cTfhPD-1-/+/hi, cTfh1,2,17 and cTfh1/17). The level of these subsets was then compared between normal subjects and patients with lymphoma. We found that the method was robust enough to obtain reliable results from routinely collected patient material. The technique we describe for the analysis can be easily adapted to cell sorting and downstream applications such as RT-PCR.

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets.

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the ...

Biology

Magnetic Activated Cell Sorting (MACS): Isolation of Thymic T Lymphocytes

Source: Meunier Sylvain1,2,3, Perchet Thibaut1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
Defence against pathogens depends on surveillance by the immune system. This system is complex and comprises many cell types, each one with specific functions. This complex composition enables immune responses to a large diversity of pathogens and injuries. Adaptive immunity allows specific responses against specific pathogens. The majority of cells responsible for this type of immunity are the lymphocytes (B cells and T cells). Usually, B cells respond to extracellular infections (such as bacterial infections), and T cells respond to intracellular infections (such as viral infections). The different types of cells in lymphocyte populations can be characterized by the combination of cell surface proteins they express and/or by a panel of secreted cytokines.
Magnetic sorting allows enrichment of targeted cell populations using magnetic properties and expression of one or several cell surface proteins (1, 2). This technique consists of three steps. First, the cells are incubated with magnetic beads that are coupled with one or several monoclonal-specific antibodies. Cells that express surface proteins that bind to these antibodies attach to the magnetic beads. Then, the targeted cell populations are captured with a magnet. To finish, the targeted cells are eluted from the magnet. At the end, two sorting products are obtained, one containing unlabeled cells and the second containing the target cells coupled with the magnetic beads. Columns can be used to improve the efficiency of magnetic sorting. In the column, a non-magnetic element lengthens the path of cell through the column. Hence, cell flow is slowed down, facilitating cell capture by the magnet.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10495/10495fig1.jpg" /> 
Figure 1: Schematic representation of magnetic separation. Thymic leukocytes are stained with anti-CD3 biotinylated antibodies. After washing, streptavidin (SAV) coupled beads specifically fix the biotin on anti-CD3 antibodies. (1) Cells are transferred in a column. (2) The magnet does not retain unlabeled cells, while CD3-positive cells remain in the column. Finally, the column is separated from the magnet and (3) CD3-positive cells are eluted in medium. <a href="https://www.jove.com/files/ftp_upload/10495/10495fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
There are two types of magnetic sorting (3). In positive sorting, cells of interest are captured with the magnetic beads. In negative sorting, unwanted cells are removed by capturing with the magnetic beads carrying the appropriate antibodies. This MACS technique permits good enrichment of targeted cells and improves the percentage of recovered cells from 1-20% to 60-98% in an organ. After sorting, it is necessary to verify cell purity and sorting by different methods (e.g. flow cytometry). The MACS technique is ideal to enrich a target population for other experiments such as cell culture or cell cycle analysis.
In this lab exercise, we demonstrate how to isolate thymic leukocytes and thereafter enrich thymic CD3-positive cells from the mix using magnetic cell sorting technique.