Isolation of mouse T cells from spleen

5 posts / 0 new
Last post
Mouse
Mouse's picture
Isolation of mouse T cells from spleen

I am trying to isolate some mouse T cells from spleen. Would anyone have an inexpensive and efficient protocol for achieving this? What purity can I expect to achieve with the given protocol?

Tony Rook
Tony Rook's picture
Mouse -

Mouse -

There a several commercially available kits on the market today. Here is a summary of these and links to product info/literature...

R&D Systems - MagCellect* Mouse CD3+ T Cell Isolation Kit (Catalog Number: MAGM201)

The kit contains sufficient reagents to process 1x10^9 total cells.

Intended Use
MagCellect Mouse CD3+ T Cell Isolation Kit is designed to isolate CD3+ T cells via a negative selection principle. The resulting cell preparation is highly enriched with CD3+ T cells. Typical recovery ranges between 45% and 65% and the purity of recovered CD3+ T cells ranges between 90 and 98%.

Background
R&D Systems MagCellect products are designed for the isolation of cells in a liquid phase. R&D Systems MagCellect technology is based on the use of Ferrofluids or magnetic nanoparticles that have no magnetic memory (superparamagnetic). These Ferrofluids have a diameter of ~150 nm and as a result behave like colloidal particles. This feature allows the Ferrofluids to remain in solution without the need for mixing and additionally allows for efficient diffusion kinetics during the binding reaction. The proprietary manufacturing technology of MagCellect Ferrofluids generates particles with higher ligand binding capacity per mass compared to many other larger diameter magnetic particles.

Principle of Selection
A mononuclear cell suspension is first incubated with the MagCellect Antibody Cocktail which targets the unwanted cells. MagCellect Streptavidin Ferrofluid is next added to the reaction which allows the streptavidin coated nanoparticles to interact with biotinylated antibody tagged cells. The tube containing the cell suspension is then placed within a magnetic field. Magnetically tagged cells will migrate toward the magnet (unwanted cell fraction), leaving the untagged cells or desired cell population in suspension to be harvested by aspiration while the tube remains in the magnetic field. The enriched cell preparation is then available for a variety of applications including tissue culture, immune status monitoring and flow cytometry.

Cell Selection Procedure
This procedure is for the processing of 20x107 total cells using 5 mL tubes and the MagCellect Magnet.

*This product utilizes and/or contains technology licensed from Immunicon Corporation, Huntingdon Valley, Pennsylvania 19006, USA, which is covered by one or more claims of United States and International patents and/or pending patent applications.

___________________________________________________

Dynal Biotech (Invitrogen) - Dynabeads

T Cell Isolation - Simple, Efficient and Consistent
Human and mouse T cell isolation with Dynabeads could not be easier. Add Dynabeads to your starting sample, incubate and watch as the bead-captured target cells are pulled towards the tube wall by the handy magnet. You will get a high cell yield and viability, and as you do not need columns, you will have a full recovery of cells.

Positive Isolation of T Cells
As well as being the only magnetic isolation system to isolate human T cells directly from whole blood, you can isolate from any starting sample, including bone marrow or buffy coat. Mouse cells are separated from spleen, lymph node or thymus. The supernatant is removed leaving a high yield of pure human or mouse T cells that are suitable for downstream molecular applications. With DETACHaBEAD, you can remove the Dynabeads from some cells and use antibody-free T cells for further culturing and flow cytometry.

T Cell Depletion
Perform as with positive isolation, but you discard the beads with the unwanted cells. A perfect strategy for removing activated human CD25+ T cells.

_____________________________________________________

Miltenyi Biotec MACS Separation - CD8a+ T Cell Isolation Kit

Description

The CD8a+ T Cell Isolation Kit is an indirect magnetic labeling system for the isolation of untouched cytotoxic CD8a+ T cells from single-cell suspensions of spleen and lymph nodes. Highly pure untouched CD8a+ T cells are isolated by depletion of non-CD8a+ T cells. Non-CD8a+ T cells, i.e. T helper cells, B cells, NK cells, dendritic cells, macrophages, granulocytes, and erythroid cells, are indirectly magnetically labeled with a cocktail of biotin-conjugated antibodies against CD4 (L3T4), CD45R (B220), CD49b (DX5), CD11b (Mac-1), and Ter-119, as well as Anti-Biotin MicroBeads. Isolation of CD8a+ cytotoxic T cells is achieved by depletion of the magnetically labeled cells. The CD8a+ T Cell Isolation Kit includes the Biotin-Antibody Cocktail for labeling of non-CD8+ T cells and Anti-Biotin MicroBeads.

Applications

Untouched CD8a+ cytotoxic T cells are used for functional studies where effects due to crosslinking of surface molecules should be excluded or if further magnetic separation of CD8a+ T cells into subpopulations is intended, e.g. for the isolation of naive CD8a+ T cells(1). 1 CD8a+ T cells isolated with the CD8a+ T Cell Isolation Kit were used for adoptive transfer to evaluate the migration and proliferation properties of transferred T cells and to evaluate how to increase the function of low-avidity tumor-specific T cells in mouse tumor models(2,3). Isolated CD8a+ T cells were also used to analyze the role of OX40 for the survival of primed CD8 T cells after adoptive transfer into tumor-challenged mice(4). In addition, CD8a+ T cells were isolated from tumor tissues and further analyzed for their cytotoxicity(5)

MACS References

1. Gruber et al. (2005) 175: 201-206.[7317]
2. Yu et al. (2004) Nat. Immunol. 5: 141-149.[4309]
3. Lyman et al. (2005) 174: 2563-2572.[6789]
4. Song et al. (2005) J. Immunol. 175: 3534-3541.[7288]
5. Fan et al. (2006) Blood 107: 1342-1351.[8423]

___________________________________________________

Miltenyi Biotec MACS Separation - CD4+CD25+ Regulatory T Cell Isolation Kit

Description

The CD4+CD25+ Regulatory T Cell Isolation Kit was developed for the isolation of CD4+CD25+ regulatory mouse T cells from single-cell suspensions of spleen and lymphnodes.
CD4+CD25+ immunoregulatory T cells have been shown to actively suppress immune responses against autologous and foreign antigens in vivo and in vitro. CD25, the IL-2Rα chain, is also expressed on activated CD4+ T cells, CD8+ T cells, dendritic cells, and B cells.

The kit contains a cocktail of lineage specific biotin-conjugated antibodies against CD8 (Ly-2), CD11b (Mac-1), CD45R (B220), CD49b (DX5), Ter-119, and Anti-Biotin MicroBeads for depletion of non-CD4+ T cells, as well as CD25-PE and Anti-PE MicroBeads for subsequent positive selection of CD4+CD25+ regulatory T cells.

Applications

Regulatory T cells isolated from mouse lymph nodes with the CD4+CD25+ Regulatory T Cell Isolation Kit were used in co-culture experiments with dendritic cells to study priming of dendritic cells for tolerance induction in vitro and after adoptive transfer of primed dendritic cells in vivo(1). Furthermore, they were isolated from UV-exposed mice for adoptive transfer experiments. In this context, the role of regulatory T cells for induction and elicitation of hapten-specific tolerance was analyzed(2). Isolated regulatory T cells were also used for adoptive transfer in an experimental autoimmune thyroiditis mouse model, where they were shown to suppress a thyroglobulin-specific autoimmune response(3), as well as in a melanoma mouse model where they prevented tumor immunity(4). In addition, in vitro suppression assays were performed with isolated regulatory T cells in studies e.g. analyzing the effects of signaling molecules on the regulation of regulatory T cell development(5) or analyzing the effect of injury on regulatory T cell function in a mouse burn injury model(6).

MACS References

1. Fallarino et al. (2003) Nat. Immunol. 4(12): 1206-12.[3982]
2. Schwarz et al. (2004) J. Immunol. 172: 1036-1043.[3791]
3. Gangi et al. (2005) J. Immunol. 174: 7006-7013.[7184]
4. Turk et al. (2004) J. Ex. Med. 200(6): 771-782.[6334]
5. Kashiwada et al. (2006) J. Immunol. 176: 3958-3965.[8594]
6. Choileain et al. (2006) J. Immunol. 176: 225-236.[9411]

___________________________________________________

StemCell Technologies Inc

StemCell offers four (4) kits for T-cell isolation...

EasySep Mouse CD90 (Thy1.2) Positive Selection Kit

Recommended for:
These reagents are designed to positively select CD90+ (Thy1.2) cells (cells expressing the CD90 antigen) from single cell suspensions of spleen or other tissues from mouse strains expressing Thy1.2. CD90 is expressed on most T cells but not B cells.

SpinSep Mouse T Cell Enrichment Cocktail

Recommended for:
This depletion cocktail is tailored to highly enrich T cells from suspensions of murine spleen cells. The recovered T cells have not been labeled with antibody.

EasySep Mouse T Cell Enrichment Kit

Recommended for:
This depletion cocktail is tailored to highly enrich T cells from suspensions of murine spleen cells. The recovered T cells have not been labeled with antibody.

StemSep Mouse T Cell Enrichment Kit

Recommended for:
This depletion cocktail is tailored to highly enrich T cells from suspensions of mouse spleen cells. The recovered T cells have not been labeled with antibody.

____________________________________________________

I hope one of these kits can help....

Good luck!

Tony Rook
Tony Rook's picture
Here are some references that

Here are some references that may be of some use to you....

Joseph S. Giglia, Geraldine M. Ovak, M. A. Yoshida, Carol J. Twist, Amy R. Jeffery and John L. Pauly. Isolation of Mouse T-Cell Lymphoma Lines from Different Long-Term Interleukin 2-dependent Cultures. Cancer Research 45, 5027-5034, October 1, 1985

Abstract:

A number of different biological properties have been ascribed to the hormone-like protein interieukin 2 (IL-2). However, the most salient feature of this lymphokine is its ability to sustain the long-term proliferation of T-cells from humans and mice. Reported herein are the results of studies demonstrating the isolation of growth factor-independent cell lines from the long-term IL-2-dependent murine T-cell line CTLL-2 that is used frequently as the source of target cells in IL-2 bioassays. Sustained logphase growth of these T-cells in vitro has been achieved using Retri dishes of polymethylpentene; growth could not be sustained in similar dishes of glass, untreated polystyrene, polystyrene that had been treated for cell culture, or polycarbonate.

The IL-2-independent line grew as a T-cell lymphoma when injected i.p. into pristane-treated, but not untreated, syngeneic C57BL/6 mice. In contrast, cells from the IL-2 parental line CTLL- 2 did not grow in vivo.

Characterization of the IL-2-independent lines propagated in vitro (denoted as line CEC) or in vivo (denoted as line CEP) demonstrated that they retained their dependency for 2-mercaptoethanol and expressed phenotypic profiles of their parental line CTLL-2 (Thy 1.2+, Lyt-1-; Lyt-2-). Isolation of an IL-2-independent T-cell lymphoma from a CTLL-2 line obtained from another investigator using a protocol that has proven reproducible under carefully controlled laboratory conditions and defined phenotypic traits of the syngeneic T-cell isolates provided evidence that the tumors were not a cross-culture contaminant arising as a result of a laboratory accident. Moreover, karyotypic analysis using a quinacrine: Hoechst banding technique revealed similar marker chromosomes in the IL-2-dependent and -independent lines.

IL-2-independent lines have also been established from the IL-2-dependent murine T-cell line CT-6.

Accordingly, the results of these studies suggest that, during prolonged cultivation that has included exposure to crude IL-2 preparations known to contain phorbol ester, possibly viruses, and other contaminants, the IL-2-dependent lines have developed subpopulations that are thought to have undergone malignant transformation of unknown etiology to generate IL-2-independent murine T-cell lymphomas that can be passaged repetitively either in vitro or in vivo.

______________________________________________________

Shengkun Suna, Zikuan Guoa, Xuren Xiao, Bing Liu, Xiaodan Liu, Pei-Hsien Tang, Ning Mao. Isolation of Mouse Marrow Mesenchymal Progenitors by a Novel and Reliable Method. Stem Cells 2003;21:527-535

Abstract:
Bone marrow contains a population of rare progenitor cells capable of differentiating into osteoblasts, chondrocytes, adipocytes, myoblasts, and hematopoiesis-supporting stromal cells. These cells, referred to as mesenchymal progenitor cells (MPCs), can be purified and culture-expanded from animals and humans. Using bone-marrow-conditioned medium combined with basic fibroblast growth factor, we cultured a relatively homogeneous population of MPCs from murine bone marrow, which uniformly expressed stem cell antigen-1, CD29, CD44, c-kit, and CD105, while being negative for expression of CD45, CD31, and CD34. In vitro differentiation assays showed the tripotential differentiation capacities of these cells toward adipogenic, osteogenic, and chondrogenic lineages. Most importantly, immunophenotypic analyses demonstrated that MPCs did not express major histocompatibility complex class II molecules or the T-cell costimulatory molecules CD80 and CD86, consistent with further investigation showing that MPCs failed to elicit a proliferative response from allogeneic lymphocytes. Moreover, when allogeneic or third-party MPCs were added to T cells stimulated by allogeneic lymphocytes or the potent T-cell mitogen concanavalin-A, a significant reduction in T-cell proliferation was observed. In conclusion, our data demonstrate that we successfully isolated and culture-expanded a relatively homogeneous population of MPCs from adult murine bone marrow. Additionally, these primary cells could suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. This immunoregulatory feature of MPCs strongly implies that they may have potential applications in allograft transplantation.

________________________________________________________

NE Street, JH Schumacher, TA Fong, H Bass, DF Fiorentino, JA Leverah and TR Mosmann. Heterogeneity of mouse helper T cells. Evidence from bulk cultures and limiting dilution cloning for precursors of Th1 and Th2 cells. The Journal of Immunology, Vol 144, Issue 5 1629-1639.

Abstract:
Many long term mouse Th clones express either the type 1 or type 2 Th cell (Th1 or Th2) cytokine secretion phenotype. In this report we present two lines of evidence for the existence of additional Th differentiation states. Lectin-stimulated spleen cells secreted moderate levels of IL-2 compared with long term Th1 clones, whereas the levels of other cytokines were more than 100-fold lower than those produced by either Th1 or Th2 clones. This suggests that many spleen cells produce substantial amounts of IL-2 but little or no IL-4, IL-5, IFN-gamma, IL-3, and granulocyte/macrophage-CSF. In contrast to long term Th clones, many short term alloreactive clones displayed cytokine secretion phenotypes intermediate between the Th1 and Th2 patterns. The proportion of recognizable Th1 and Th2 clones at early times in culture was greatly increased by immunization of the mice from which the responder and stimulator cells were derived; Brucella abortus immunization resulted in the isolation of exclusively Th1 clones, whereas infection with Nippostrongylus brasiliensis resulted in a strong trend toward the isolation of Th2 clones. The immunization of mice from which responder cells were derived strongly affected the type of Th clone obtained, whereas the source of stimulator cells had much less effect, suggesting that the commitment of Th cells to the Th1 or Th2 phenotypes occurred mainly in vivo. A model for the possible relationships of the various Th cells is presented.

________________________________________________________

D Vremec, M Zorbas, R Scollay, DJ Saunders, CF Ardavin, L Wu and K Shortman. The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells. Journal of Experimental Medicine, Vol 176, 47-58.

Abstract:
A new procedure for rapid isolation of dendritic cells (DC) was devised, involving collagenase digestion of tissues, dissociation of lymphoid-DC complexes, selection of light-density cells, then depletion of lymphocytes and other non-DC by treatment with a mixture of lineage- specific monoclonal antibodies (mAbs) and removal with anti- immunoglobulin-coupled magnetic beads. This enriched population (approximately 80% DC) was further purified when required by fluorescence-activated cell sorting for cells expressing high levels of class II major histocompatibility complex (MHC). The isolated DC were characterized by immunofluorescent staining using a panel of 30 mAbs. Thymic DC were surface positive for a number of markers characteristic of T cells, but they were distinct from T-lineage cells in expressing high levels of class II MHC, in lacking expression of the T cell receptor (TCR)-CD3 complex, and having TCR beta and gamma genes in germline state. Splenic DC shared many markers with thymic DC, but were negative for most T cell markers, with the exception of CD8. A substantial proportion of DC from both thymus and spleen expressed CD8 at high levels, comparable with that on T cells. This appeared to be authentic CD8, and was produced by the DC themselves, since they contained CD8 alpha mRNA. Thymic DC presented both the CD8 alpha and beta chains on the cell surface (Ly-2+3+), although the alpha chain was in excess; the splenic DC expressed only the CD8 alpha chain (Ly-2+3-). It is suggested that the expression of CD8 could endow certain antigen- presenting DC with a veto function.

_________________________________________________________

Fabienne Anjuère, Pilar Martín, Isabel Ferrero, Marta López Fraga, Gloria Martínez del Hoyo, Natalia Wright, and Carlos Ardavín. Definition of Dendritic Cell Subpopulations Present in the Spleen, Peyer's Patches, Lymph Nodes, and Skin of the Mouse. Blood, Vol. 93 No. 2 (January 15), 1999: pp. 590-598

Abstract:
Dendritic cells (DC) are highly efficient antigen-presenting cells (APC) that have an essential function in the development of immune responses against microbial pathogens and tumors. Although during the past few years our understanding of DC biology has remarkably increased, a precise characterization of the different DC subpopulations remains to be achieved with regard to their phenotype and lineage relationships. In this report, we have extensively studied the DC subpopulations present in the thymus, spleen, Peyer's patches, lymph nodes (LN) and skin of the mouse. Thymus DC and 60% spleen DC have a lymphoid DC phenotype, ie, CD8+ DEC-205high Mac-1low, whereas 40% spleen DC have a myeloid DC phenotype, ie, CD8- DEC-205low Mac-1high. Both CD8+ and CD8- DC are leukocyte function-associated antigen-1 (LFA-1)high and highly adherent. Within Peyer's patches the majority of DC correspond to the CD8+ DEC-205high Mac-1low lymphoid category. In the LN, together with CD8+ and CD8- DC, an additional nonadherent CD8int LFA-1int subpopulation with lymphoid DC characteristics is described. Finally, in the skin both epidermal Langerhans cells (LC) and dermal DC are CD8-DEC-205high Mac-1 high , and do not express LFA-1. Interestingly, LC migration experiments indicate that LC underwent the upregulation of CD8 and LFA-1 upon migration to the LN, supporting the hypothesis that LC belong to the CD8+ lymphoid lineage.

_______________________________________________________

Invitrogen Customer Report. Rebecca B. Blank - Department of Pathology, Tropical Disease Research Unit, University of California, San Francisco

Isolation of an Untouched CD4+ T Cell Population from bothMurine Spleen and Peripheral Lymph Nodes using Dynal MouseCD4 Negative Isolation Kit.

Abstract:
The isolation of an untouched CD4+ T cell population is desirable for many applications in immunology research including in vitro effector and memory cell function assays and cytokine production assays. Previous methods using CD4 antibody coated magnetic beads for positive isolation of CD4+ T cells produced rapid isolation of a pure population but detachment of the bead
from the cell was often necessary before CD4+ T cells could be further analyzed in downstream applications such as in vivo reconstitution experiments.
The introduction of the Dynal Mouse CD4 Negative Isolation Kit allows for high yield and high purity isolation of CD4+ T cells without the requirement of bead detachment and with the
confidence that the isolated CD4+ T cell population remains untouched and unaltered by the isolation process.
Here we describe the results of two negative isolation experiments with the Dynal Mouse CD4 Negative Isolation Kit in both a splenic cell population and a heterogeneous splenic and
peripheral lymph node population from uninfected C57BL/6 mice.

_______________________________________________________

Again - I hope these help and Good Luck!

Mouse
Mouse's picture
Thanks for the information. I

Thanks for the information. I am fully aware of the magnetic bead separation methods. However, due to the normal funding issues, I was hoping someone would have an inexpensive and efficient protocol for isolation of mouse T cells. More to the tune of a simple homogenization and using cotton wool. However, I have just briefly read about this and was hoping to get a protocol and some tips.

We normally use human PBMC and get our T cells through mitogenic stimulation in vitro. We would now like to extend our studies to mouse T cells and perform some ex vivo modifications with them.

Thanks eitherway for the information. Alot of it was on DC's which I am not working on.

Tony Rook
Tony Rook's picture
Here is a reference which

Here is a reference which uses the cotton-wool isolation technique you mentioned. They report a 95% recovery efficiency.

"Immunization of Mice.--A/J mice were inoculated intraperitoneally with 0.5 mg of KLH in 0.2 ml of Freund's complete adjuvant. Spleens were removed 2-3 wk after the injection. Single-cell suspensions were prepared by teasing the cells through a lO0-mesh stainless steel screen in the presence of medium. Mter washing once with medium, the cells were passed through a column containing 0.1 g of cotton wool per 108 cells, then washed three times with medium. More than 95% of the leukocytes present had the morphology of small lymphocytes."

Reference:

TAT K. CHOI, DOUGLAS R. SLEIGHT, AND ALFRED NISONOFF. GENERAL METHOD FOR ISOLATION AND RECOVERY OF B CELLSBEARING SPECIFIC RECEPTORS. THE JOURNAL OF EXPERIMENTAL MEDICINE, 1974. VOLUME 139, 761-766.

Abstract:
Starting with the work of Wigzell and Andersson (1) a number of methods have been described for the isolation of cells bearing specific receptors. Depletion of a particular set of cells has been accomplished with a variety of immunoadsorbents. Recovery of cells is a more complex problem. As shown by Wofsy et al., cells with antihapten receptors can be recovered in a viable state by elution with free hapten at neutral pH (2). A more general method consists in adsorbing specific populations of cells onto Sephadex beads to which antigen is covalently conjugated. The Sephadex is then digested with dextranase to liberate the cells (3). Edelman and his collaborators adsorb cells onto nylon
strings to which the antigen is covalently linked and recover the cells by plucking the strings. The mechanism may involve either the removal of receptors from cells under the mechanical stress or the breaking of noncovalent bonds linking the cell receptor to the antigen. The ingenious cell sorter devised by Herzenberg and his associates pro~cides an effective general technique for
depletion or recovery of a specific population of cells (5). The instrument is, however, quite expensive. Cells obtained by this method necessarily have some fluorescent antigen, or fluorescent antibody directed to a cell-surface antigen, on their surfaces; this does not appear to affect their utility for many
biological experiments.

We report here a method, carried out at neutral pH, which may be generally applicable to the isolation of B cells bearing specific receptors. In principle, the method consists in coating polystyrene tubes (6) with specifically purified rabbit anti-p-azobenzoate antibody. This is followed by an anfigen-azobenzoate conjugate; a few micrograms are bound. Cells with receptors for the antigen are then bound specifically to the tube, and are released upon addition of free hapten. Only a small fraction of the antigen-azobenzoate conjugate is
released under conditions which suffice to permit recovery of nearly all of the cells.

References cited in the abstract:

1. Wigzell, H. and B. J. Andersson. 1969. Cell separation on
antigen-coated columns. J. Exp. Med. 129:23.
2. Wofsy, L., J. Kimura, and P. Truffa-Bach. 1971. Cell
separation on affinity columns: the preparation of pure
populations of anti-hapten specific lymphocytes. J. Immunol.
107:725.
3. Schlossman, S. F., and L. Hudson. 1973. Specific purification
of lymphocyte populations on a digestible immunoabsorbent.
J. Immunol. 110:313.
4. Edelman, A. M., V. Rutishauser, and C. Millette. 1971. Cell
fractionation and arrangement on fibers, beads, and surfaces.
Proc. Natl. Acad. Sci. U. S. A. 68:2153.
5. Julius, M. H., T. Masuda and L. A. Herzenberg. 1972.
Demonstration that antigen-binding cells are precursors of
antibody-producing cells after purification with a
fluorescence-activated cell sorter. Proc. Natl. Acad. Sci. U. S.
A. 69:1934.
6. Catt, K., and G. W. Tregear. 1967. Solid-phase
radioimmunoassay in antibodycoated tubes. Science (Wash.
D. C.). 158"1570.