Name: isolation, identification, and purification of murine thymic epithelial cells
Uploaded: 2014-08-08T15:00:00.000+00:00
Duration: 7 min 20 s
Description: Watch this Scientific Journal Video about Isolation, Identification, and Purification of Murine Thymic Epithelial Cells at JoVE.com

This work was supported by National Institutes of Health Grant R01 AI088209 (to K.A.H.). We also thank the University of Minnesota Flow Cytometry Resource.

In this study, adult (6 - 8 weeks) female C57Bl/6 mice were used. Mice were purchased from the National Cancer Institute and maintained under specific pathogen free conditions. The University of Minnesota Institutional Animal Care and Use Committee (IACUC) approved all animal experimentation.
1. Preparation of Instruments and Buffers
<ol>
	<li>Prepare enzyme solution (RPMI1640 medium with 0.05% [w/v] of Liberase TH and 100 U/ml of DNase I), albumin-rich buffer (1X PBS [Ca2+/Mg2+-free] with 0.5% Bovine serum albumin and 2 mM Ethylenediamine Tetraacetic acid [EDTA]), FACS buffer (1X PBS [Ca2+/Mg2+-free] containing 1% Fetal bovine serum [FBS] and 0.02% NaN3), FACS sorting buffer (1X PBS [Ca2+/Mg2+-free] containing 2% FBS), and RP10 medium (RPMI-1640 medium supplied with 10% FBS).</li>
	<li>Prepare 10 panning plates. Prepare 40 ml coating solution (0.01 mg/ml Goat anti-Rat IgG in 0.1 M NaHCO3 Buffer), and add 4 ml of coating solution into each 100 x 15 mm plate, swirl and incubate at 4 &#176;C O/N. Pour off antibody solution and rinse the plate using 5 ml of 1x PBS for 2 times, swirl vigorously between rinses. Store the prepared plates at 4 &#176;C.</li>
</ol>
2. Harvest of Thymus Tissue
<ol>
	<li>Euthanize mouse in a CO2 chamber. Put the animal on its back on a dissection mat, pin the feet on the mat and wet the fur by spraying with 70% ethanol.</li>
	<li>Make a midline incision through the skin, starting from above the urethral opening straight up to the lower jaw with scissors and extend the incision downward to the knees on both sides. The incision looks like an upside down &#8220;Y&#8221;. Pull the loose skin back on the sides, and pin it to the dissection mat.</li>
	<li>Puncture the diaphragm from the xiphoid, cut the ribs on each side up to about the clavicle, then lift up the ribs with forceps and pin it to the dissection mat. The thymus is just under the ribs, and looks like two thin white lobes overlying the heart (Figure 1). Disconnect connective tissue surrounding the thymus, gently pull and remove the pair of thymus lobes with curved serrated forceps. Place thymus lobes into a 6 well plate containing 5 ml RPMI-1640.</li>
</ol>
3. Preparation of Thymic Stromal Cells
<ol>
	<li>Trim any surrounding fat and connective tissue and transfer the thymus lobes to a new well of the 6 well plate containing 5 ml freshly prepare enzyme solution.&#160; Make small incisions in the thymus lobes with fine scissors, and incubate the plate at 37 &#176;C for 20 min.</li>
	<li>Gently pipette digesting thymus tissue up and down 2 - 3 times using 5 ml pipette for tissue dissociation. Collect supernatant fraction in a 50 ml tube containing 10 ml cold albumin-rich buffer to neutralize enzymes.&#160; Keep the collection tube on ice.&#160; Add 2.5 ml enzyme solution to the remaining tissue and incubate the plate at 37 &#176;C for 15 min.</li>
	<li>Perform gentle mechanical agitation with a 3 ml syringe and 18 G needle to break up aggregates.&#160; Pool supernatant into the collection tube.&#160; Add 2.5 ml enzyme solution to the remaining tissue and incubate the plate at 37 &#176;C for 15 min.</li>
	<li>Repeat gentle mechanical agitation with a 3 ml syringe and a 25 G needle, and incubate the plate additional 5 - 10 min.</li>
	<li>After complete digestion, pool the supernatant into the collection tube.&#160; Centrifuge the collection tube at 400 x g, 4 &#176;C for 8 min to collect the cells.&#160; Aspirate the liquid and suspend the pelleted cells in 10 ml albumin-rich buffer and filter the sample through 100 mm mesh.&#160;&#160; Count cells using a hemocytometer.&#160; Then perform flow cytometic analysis (Section 4) or TECs enrichment (Section 5).</li>
</ol>
4. Identification of TECs by Flow Cytometry
<ol>
	<li>Prepare cells to a final concentration of 4 x 106 cells per 100 &#181;l in FACS buffer for staining in a 96 well round-bottom test plate and keep the plate on ice. Add 20 &#181;l Fc block solution (anti-CD16/CD32 antibody at 10 &#181;g/ml in FACS buffer) to the cells and incubate the cells for 10 min on ice. Spin the plate at 400 x g, 4 &#176;C for 3 min. Discard liquid from wells by flicking the plate face down into a sink.</li>
	<li>Suspend the pelleted cells in 100 &#181;l antibody cocktail (anti-CD45, -EpCAM, -MHC class II, and -Ly51 antibodies, and the UEA-1 lectin labeled with different fluorochromes at manufacturer recommended or tested optimal concentrations of each antibody in FACS buffer), and incubate the cells on ice for 20 min in the dark. For compensation, stain 1 x 106 cells with each individual fluorochrome.</li>
	<li>Add 100 &#181;l of FACS buffer to each well and spin the plate at 400 x g, 4 &#176;C for 3 min. Repeat the wash step one time, and then resuspend cells in 100 &#181;l FACS buffer. Transfer the cells to a 12 x 15 mm round-bottom FACS tube filled with 300 &#181;l FACS buffer. Acquire samples on flow cytometer and analyze data using FACS analysis software. TEC gating strategy is shown in Figure 2.</li>
</ol>
5. Enrichment of TECs by Panning
<ol>
	<li>Spin down the thymus cells (prepared in Section 3) at 400 x g, 4 &#176;C for 5 min. Aspirate the liquid and suspend the pelleted cells in FACS sorting buffer at a concentration of 2 x 108 cells per ml in a 15 ml or 50 ml conical tube. Add anti-CD90.2 antibody (Clone 30-H12) (or anti-CD45) at a final concentration of 2.5 &#181;g/ml of cell suspension and incubate cells gently on a rotating mixer at 4 &#176;C for 30 min. Following the incubation, wash with a 5 - 10x volume of RP10 medium and centrifuge the tube at 400 x g, 4 &#176;C for 5 min.</li>
	<li>Aspirate the liquid and resuspend the pelleted cells in RP10 medium at a concentration of 1 x 107 per ml. Add 5 ml cell suspension to each coated panning plate (prepared in Step 1.3), swirl and incubate for 30 min at RT. Swirl vigorously; transfer the supernatants into a conical tube. Rinse the plate two times using RP10 medium and pool the supernatants into the collection tube.</li>
	<li>Centrifuge the collection tube at 400 x g, 4 &#176;C for 5 min. Aspirate the liquid and resuspend the pelleted cells in 5 ml RP10 medium. Add the cell suspension to a new panning plate, swirl and incubate for 30 min at RT. Collect the cells rinse the plate and pool them into a conical tube. Once TECs are enriched, spin down the cells at 400 x g, 4 &#176;C for 5 min and suspend cells in 5 ml FACS sorting buffer.</li>
</ol>
6. Purification of TECs by Cell Sorting
<ol>
	<li>Count cells using a hemocytometer and prepare cell suspension at a concentration of 4 x 107 cells in FACS sorting buffer. Add antibodies described in Section 4.4 into cell solution and incubate cells gently on a rotating mixer at 4 &#176;C for 30 min. Following staining, wash and suspend cells to a concentration of 10 x 106 cells per ml of FACS sorting buffer.</li>
	<li>Sort TEC subsets through a 100 &#181;M nozzle using fluorescence activated cell sorting. Sorted cell are collected in 30% (v/v) FBS in RPMI-1640). Once cell sorting is done, centrifuge cells at 400 x g, 4 &#176;C for 5 min and prepare for downstream analysis.</li>
</ol>

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The authors declare that they have no competing financial interests.

In the protocol, critical steps are the preparation of thymus stromal cells (Section 3) and the enrichment of TECs (Section 4). It is strongly recommended that fresh enzyme solution is prepared each time and the tissue is treated as soon as possible. For pooled thymi, optimizing the volume of enzyme solution is required depending upon the number of thymi. If any tissue residue is left after the processing, adding more enzyme solution and extension of incubation time could increase cell yield. During enrichment of TECs, completing collection of the supernatant and repeating the plate rinse outlined in Section 5.3 will decrease cell loss.
In 1980, Wekerle et al. first reported a type of TECs - &#8221;thymic nurse cells&#8221; (TNCs)33, which are large cortical epithelial cells that completely envelop many viable lymphoid cells within intracellular vesicles. In 1982, Kyewski and Kaplan described various isolation conditions that influence the yield of TNCs34. Recently, Takahama and colleagues revisited the isolation and characteristics of TNCs. They identified such cells on the basis of preferential staining for CD45 only after membrane permeabilization35. They also described &#8220;broken&#8221; TNC in their preparation, which consisted of a cTEC and multiple bound thymocytes. Such broken TNC were positive for extracellular CD45 and for the cTEC marker &#946;5t and comprised 70% of total cTECs in their preparation. Indeed, we also observed a similar cell population, which was CD45high EpCAM+ and Ly51+ in our prepared thymus cell suspension, although comprised less than 40% of total cTECs with our isolation procedure. These broken TNC are removed from TEC enrichment protocols, like ours, that utilize depletion with either CD90 or CD45. Thus, a future challenge will be to minimize the loss of this population.
Compared to the existing multi-step digestion procedure29, the Liberase digestion protocol allows a significantly higher cell yield of TECs (Figure 3). Other groups have also reported their new methods for thymic stromal cell isolation by modifying Gray&#8217;s method30,36, although cell yield was not significantly increased. Recently, Chidgey and colleagues independently reported a TEC protocol using Liberase digestion and they also obtained effective release of TECs37. However, the first thymocyte release step of their protocol has the potential to discard about 1.6 x 104 TECs from each thymus29, most of which are cTECs. Due to density gradient enrichment causing loss of smaller stromal cells, an AutoMACS-based CD45-microbead depletion is popularly used for enrichment of TECs prior to FACS purification29,36,37. Compared to the AutoMACS-based enrichment using CD45-microbead depletion protocols29, the panning enrichment obtained higher recovery rates of TECs. Overall, the protocol presented here aggregates a number of previously reported advantages. TEC subsets purified with this protocol (Section 6) have been successfully used for further analysis, such as Real Time-PCR and Western blot analysis12.
This protocol is also suitable for isolation and study of other thymic stromal cells, such as dendritic cells. We believe that robust isolation of thymic stromal cells, particularly TECs, will accelerate understanding of how the thymus functions for T cell development and achieving in vitro T cell constitution. Furthermore, characterization of these cells has great practical value for the medical community, since a common problem with recovery from therapies (such as those used in transplantation and cancer treatment) is poor reconstitution of epithelial cells in the thymus leading to immunodeficiency.

Early in T cell development, bone marrow hematopoietic stem cell-derived multipotent progenitors are recruited to the cortex of the thymus, undergo commitment to T lineage and become T cell precursors1. In the cortex, T cell precursor CD4 and CD8 double negative (DN) thymocytes expand and differentiate into immature CD4 and CD8 double positive (DP) thymocytes, forming a large pool of progenitors with highly variable T cell receptors1. Only a select MHC-restricted subset of DP cells will become CD4 or CD8 single positive (SP) thymocytes, migrate to the medulla of the thymus, and differentiate into functionally competent mature T cells, an event that is referred to as positive selection2-6. In contrast, clones of auto-reactive thymocytes undergo negative selection and are removed via apoptosis, converted to regulatory T cells for self-tolerance, or diverted to intraepithelial lymphocytes for purposes that are not yet clear3,7-10.
In the thymus, thymic stromal cells form a unique microenvironment providing signals for these various T cell development fates5,11,12. Thymic stromal cells are composed of thymic epithelial cells (TECs) - including cortical TECs (cTECs) and medullary TECs (mTECs), dendritic cells, macrophages, fibroblasts, endothelial cells, neural crest-derived pericytes and other mesenchymal cells13-15. Among these, TECs are crucial at the various stages of T cell development1,2,16,17. However, lack of a robust way to isolate TECs has hampered a comprehensive understanding of their functions16. In particular, cTECs, which form a three-dimensional network surrounding progenitors in the cortex, are essential for positive selection13,18,19 for reasons that are not yet clear. Earlier studies provided clues as to the heterogeneity and role of TECs, mostly relying on morphological and histological tools13. Recently the unique roles of TEC subsets were addressed by genetic approaches in mouse models12,20. A robust and reproducible way to isolate TECs is fundamental to achieve unbiased characterization of TEC subsets, quantitative and qualitative assessment of TEC functions, and clarification of mechanisms how cTECs support positive selection.
Due to the rarity of TECs in the thymus and the tight interactions they form in the intact organ, the isolation of TECs has been challenging. The protocol described here is based on previous discoveries, currently available reagents, techniques, and knowledge of thymus structure and stromal composition. Nearly two decades ago, several procedures were reported to disaggregate thymus tissues21-27, in which different enzymes were used during digestion, including Trypsin, Collagenase and Dispase. Gray et al. compared those enzymes in their precedure28, and reported an improved method with a multiple-step digestion of enzyme cocktails29 that became widely used20,30. However, this method involves a long preparation time and complex digestion steps and results in variable final cell numbers and proportions of TECs even in the same murine cohort29,30. Several years ago, Liberase research grade enzyme, containing highly purified Collagenase and neutral protease started to be used in thymus tissue dissociation30. Here, we describe a Liberase digestion-based protocol, with optimized mechanical separation procedures, that yields a high number of viable TECs from mouse thymus tissues.
To enrich dissociated stromal cells, previous studies have used either density gradients or magnetic bead separation21,29. However, both methods cause severe lost of certain populations of thymic stromal cells, particularly the population of cTECs29. Cytotoxic elimination and panning techniques31,32 have widely been used for depletion or separation of lymphocytes in the immunological field12,31. After comparison of these techniques, we established the current panning protocol for enrichment of TECs. The gentle condition during the enrichment procedure leads to less cell death and unbiased and increased TEC recovery.
The isolated thymus cell suspension described in Section 3 can be directly applied to flow cytometric analysis for identification and characterization of TEC subsets and dendritic cells. Section 4 describes a simple and useful way to identify TEC subsets using multiparameter flow cytometer. For experiments that seek to obtain purified cTECs or mTECs, TEC enrichment and cell sorting procedures can be found in Section 5 and 6.

<table><tbody><tr><td>Anti-mouse EpCAM</td><td>eBioscience</td><td>XX-5791-YY*</td><td>Clone G8.8</td></tr><tr><td>Anti-mouse MHC II</td><td>eBioscience</td><td>XX-5321-YY*</td><td>Clone M5/114.15.2</td></tr><tr><td>Anti-mouse Ly51</td><td>eBioscience</td><td>XX-5891-YY*</td><td>Clone 6C3</td></tr><tr><td>UEA-1</td><td>Vector Laboratory</td><td>FL-1061</td><td></td></tr><tr><td>Flow cytometer</td><td>BD Biosciences&amp;nbsp;</td><td>LSRFortessa</td><td></td></tr><tr><td>Flow cell sorter</td><td>BD Biosciences&amp;nbsp;</td><td>FACSAria</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>*XX varies by fluorochrome and YY varies by vial size.</td><td></td><td></td><td></td></tr></tbody></table>

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.

Using this protocol, a thymus organ was removed from an adult mouse (Section 2) and a thymic cell suspension was prepared as outlined in Section 3. The obtained cell suspension consisted of thymocytes, hematopoietic-derived stromal cells and non-hematopoietic stromal cells. CD45 is a hematopoietic pan-marker expressed on both thymocytes and hematopoietic stromal cells such as macrophages and dendritic cells. In contrast, TECs are not of hematopoietic origin and do not express CD4515. Cell staining and flow cytometric analysis were performed as outlined in Section 4.
Two types of TEC subsets, cTECs and mTECs, originate from a common bi-potent TEC precursor15. Both cTECs and mTECs express the EpCAM molecule on their surface28. Thus TECs can be gated according to EpCAM-positive and CD45-negative by flow cytometry (Figure 2A). cTECs and mTECs can be distinguished by two reagents: the Ly51 antibody that recognizes glutamyl aminopeptidase expressed by cTEC, and the lectin Ulex Europaeus agglutinin-1 (UEA-1)28 that binds to mTEC (Figure 2B). Both mTECs and cTECs express MHC class II molecules on their surface (Figure 2C and 2D). mTECs can be further classified into to mTEClow and mTEChigh depending upon the level of MHC II molecules (Figure 2D). The average number of TEC per thymus is about 9 x 105. In direct comparisons, we recovered approximately 8 times more TECs using this Liberase enzyme based protocol compared to a multi-step protocol with collagenase/dispase29 (Figure 3).
In order to decrease sorting time and increase viability of recovered stromal cells, we developed a panning method to deplete thymocytes because the thymus cell suspension consists of over 95% thymocytes. Cells were incubated with anti-CD90 antibody and captured by precoated plates. Compared to that in the whole prepared thymus cells (pre-enriched), the proportion of TECs was increased from less than 0.5% to over 15% in post-enriched cells (Figure 4A and 4B). Panning with anti-CD45 can also be used if, for example, one does not need to recover dendritic cells as well. In this case, TEC enrichment increases to about 80% (data not shown). After stromal cell enrichment by panning, the proportions of TEC subsets were not altered (Figure 4C and 4D), suggesting that one subset or the other is not selectively lost during panning. Compared to the pre-enrichment sample, the recovery rate of TECs was about 85% (Figure 4E).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51780/51780fig1highres.jpg" width="500" /> 
Figure 1. The mouse thymus during dissection. The thymus is located above the heart in the anterior superior thorax. After cutting and lifting the ribs, the two white lobes of the thymus are exposed. <a href="https://www.jove.com/files/ftp_upload/51780/51780fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51780/51780fig2highres.jpg" width="500" /> 
Figure 2. Identification of cTEC and mTEC with flow cytometric data analysis. Stained thymus cell samples were analyzed using flow cytometry. Representative FACS profiles are shown. The numbers indicate the percentage of each population. (A)&#160;Of live cells, a gate of CD45-negative and EpCAM-positive events represents TEC cells. (B)&#160;Within the TEC gate, two populations are separated by level of Ly51 and UEA-1. cTECs have higher expression of Ly51; mTECs have higher level of UEA-1. (C)&#160;MHC II molecules are highly expressed on cTECs. (D)&#160;mTECs consists of MHC IIhigh and MHC IIlow sub-populations, which are separately referred to as mTEChigh and mTEClow. <a href="https://www.jove.com/files/ftp_upload/51780/51780fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51780/51780fig3highres.jpg" width="500" /> 
Figure 3. A liberase-based protocol allowed higher cell yields from thymus than using a multi-step collagenase/dispase protocol. Thymus cells were prepared using a previously published multi-step collagenase/dispase protocol or this liberase-based protocol. The two groups of samples were analyzed by flow cytometry. (A)&#160;Representative FACS profiles are shown. The numbers indicate the percentages of TEC cells in each group. (B) Total cell numbers of TEC per mouse thymus are shown in the bar graph. Error bars indicate the standard deviation (n &#62;3). A two-tailed, unpaired T test was performed using software Prism. P &#60;0.0001. <a href="https://www.jove.com/files/ftp_upload/51780/51780fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="3in" src="/files/ftp_upload/51780/51780fig4highres.jpg" width="300" /> 
Figure 4. Enrichment of TECs by the panning method. FACS profiles of pre- and post-enriched thymic stromal cells are shown. Prepared thymus cells were stained with anti-CD90 antibody. CD90-positive thymocytes compose over 95% of thymus cells. During the TEC enrichment procedure, thymocytes are captured and depleted on the pre-coated panning plate, thus TECs are enriched in the supernatant. (A) and (B), TEC gates are shown. The numbers show the proportion of TECs amongst total live cells in the starting population (Pre-enrichment) or after enrichment (Post-enrichment). (C) and (D), Cells within cTEC and mTEC gates are shown. The numbers display the percentages of cTEC and mTEC in the TEC populations from each group. (E)&#160;Cell numbers of cTECs and mTECs per mouse thymus are shown in the graph. Data are representative of 5 experiments. &#8220;Pre&#8221; stands for pre-enrichment sample; &#8220;Post&#8221; stands for post-enrichment sample; small horizontal lines indicate the mean. <a href="https://www.jove.com/files/ftp_upload/51780/51780fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation, Identification, and Purification of Murine Thymic Epithelial Cells at JoVE.com

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

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 ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

31.9K Views.  University of Minnesota. 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.

Video: Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Developmental Biology

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Biology

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Optimizing Stromal Cell Isolation from Thymus and Bone Marrow

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

Author Spotlight: Advancing Hematopoietic Research Using Stromal Cell Isolation for Single Cell Sequencing

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

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 ...

Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, fibroblasts, myofibroblasts, smooth muscle cells, immune cells, and the recently identified cell type, telocytes. Telocytes are unique mesenchymal cells with long cytoplasmic processes, reaching a distance of tens to hundreds of microns from the cell body. Telocytes have recently emerged as an important intestinal stem cell niche component, providing Wnt proteins that are essential for stem and progenitor cell proliferation.
Although protocols on how to isolate mesenchyme from the mouse intestine are available, it is not clear whether these procedures allow the efficient isolation of telocytes. Isolating telocytes efficiently requires special protocol adjustments that would allow dissociation of the strong cell-cell contact between telocytes and neighboring cells without affecting their viability. Here, available intestinal mesenchyme isolation protocols were adjusted to support the successful isolation and culture of mesenchyme containing a relatively high yield of viable single-cell telocytes.
The obtained single-cell suspension can be analyzed by several techniques, such as immunostaining, cell sorting, imaging, and mRNA experiments. This protocol yields mesenchyme with sufficiently conserved antigenic and functional properties of telocytes, and can be used for several applications. For example, they can be used for co-culture with mouse- or human-derived organoids to support organoid growth with no growth factor supplementation, to better reflect the situation in the original tissue.

Here, we present a protocol to isolate murine intestinal mesenchyme, including telocytes. These can be used for several applications, such as co-culture with mouse or human-derived organoids, to support growth and better reflect the situation in the original tissue.

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, ...

Medicine

Isolating Endothelial Cells from Murine Dermal Lymphatic Capillaries

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Author Spotlight: Magnetic Bead-Based Isolation of Murine Dermal Lymphatic Endothelial Cells

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Summary

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Chapters in this video

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Isolation and Activation of Murine Lymphocytes

Stromal Cell Isolation From Hematopoietic Organs

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

Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

Murine Dermal Lymphatic Endothelial Cell Isolation