Summary

Isolation und Aktivierung von murinen Lymphozyten

Published: October 30, 2016
doi:

Summary

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.

Abstract

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.

Introduction

Mature lymphocytes generally exist in the resting state if there is no pre-existing infection or inflammation in the individual. Therefore, it is important to retain the naïve status of lymphocytes during the isolation process before performing in vitro or in vivo functional assays. The key to ensuring consistent and reproducible results is to limit any unnecessary manipulation of the cells.

Magnetic cell sorting utilizes antibodies and microbeads to label cells so as to enrich the cell population of interest. With this approach, there are two purification strategies: positive enrichment and negative depletion. Positive enrichment enriches the cell population of interest using an antibody that binds to the target cells. Negative depletion, on the other hand, depletes non-target cells, leaving the cell population of interest. In our lab, we prefer negative depletion to positive enrichment because the binding of antibodies to the target cells could potentially alter cell features and behavior. In fact, many established cell surface markers suitable for the isolation of a particular cell population are also functional receptors.

Magnetic cell sorting not only yields highly pure populations of viable target cells, it is also less time-consuming and avoids the cellular stress induced by high-pressure flow used in fluorescence-activated cell sorting (FACS). By labeling the unwanted cell populations and depleting them using a magnetic separation column, we are able to perform rapid cell isolation without compromising the viability of the target cell population. In this protocol, we demonstrate the use of negative depletion strategies to purify naïve B cells or T cells.

Protocol

Alle Mäuse gezüchtet und unter spezifischen pathogenfreien Bedingungen und alle Maus-Protokolle werden in Übereinstimmung mit den Richtlinien der Institutional Animal Care und Use Committee geführt gehalten. 1. Herstellung von Puffer und Reagenzien Bereiten vollständige Roswell Park Memorial Institute (RPMI) Medium (10% hitzeinaktiviertem fötalem Rinderserum (FBS), 2 mM L-Glutamin, Penicillin (100 IU / ml) / Streptomycin (100 ug / ml), 55 uM 2-Mercaptoethanol ). Be…

Representative Results

Magnetzellreinigung von Lymphozyten erlaubt Benutzern, eine Zielzellpopulation in einer relativ kurzen Zeit zu reinigen. Mit unserem Verarmungs Protokoll konnten wir den Prozentsatz der CD8-T-Zellen (OT-I in Rekombinations-aktivierendes Gen-1 (RAG-1) -defiziente Mäuse) von 72,8% (vor Reinigung) auf 94,2% (nach der Reinigung zu erhöhen; 1A) 4,5. Diese gereinigten Lymphozyten können dann für die nachfolgende funktionelle Assays verwendet werden , um die Lymp…

Discussion

In diesem Protokoll zeigen wir ein Verfahren für die Lymphozyten aus lymphoiden Organen zu reinigen. Zellreinigung magnetischen Kügelchen Sortieranlage verwendet, ist eine schnelle und einfache Methode, die lebensfähige, hochgereinigten Zielzellen ergibt.

Kritische Schritte im Rahmen des Protokolls

Die Lebensfähigkeit der Zellen und Zellausbeute

Die Aufrechterhaltung Lebensfähigkeit von hämatopoet…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

Die Studie wird durch das Ministerium für Bildung, Singapur (ACRF Tier1-RG40 / 13 und Tier2-MOE2013-T2-2-038) unterstützt. Das Manuskript wurde von Amy Sullivan aus Obrizus Kommunikation bearbeitet.

Materials

Materials
RPMI 1640 (without L-Glutamine) Gibco 31870025
Fetal Bovine Serum Heat inactivated 
L-glutamine Gibco 25030024
Penicillin/Streptomycin Gibco 15140114
2-mercaptoethanol Gibco 21985023
Anti-CD43 magnetic microbeads Miltenyi Biotec 130-049-801 Mix well prior use
Streptavidin microbeads Miltenyi Biotec 130-048-101 Mix well prior use
Anti-Annexin V magnetic beads Miltenyi Biotec 130-090-201 Mix well prior use
MACS LD  Miltenyi Biotec 130-042-901
96-well U-bottom sterile culture plate Greiner Bio-one 650180
96-well F-bottom sterile culture plate Greiner Bio-one 655180
100 μm cell strainer mesh To sterilize using UV radiation prior use
0.2 μm  sterile disposable filter units Nalgene 567-0020 Can be substituted with any sterile filter device
CellTrace Violet Invitrogen C34557 CTV for short; alternative to CFSE
CellTrace Yellow Invitrogen C34567 CTY for short; alternative to CFSE
CellTrace Far Red Invitrogen C34564 CTFR for short; alternative to CFSE
Cell Proliferation Dye eFluor 670 eBioscience 65-0840 CPD670 for short; alternative to CFSE
PKH26 Sigma Aldrich PKH26GL PKH26, alternative to CFSE
Name Company Catalog Number Comments
Chemicals
Dextrose Sigma Aldrich G7021
Potassium phosphate monobasic Sigma Aldrich P5655
Sodium phosphate dibasic Sigma Aldrich S5136
Phenol Red Sigma Aldrich P0290
Calcium chloride dihydrate Sigma Aldrich C7902
Potassium chloride Sigma Aldrich P5405
Sodium chloride Merck Millipore S7653 Can use from other sources
Magnesium chloride hexahydrate Sigma Aldrich M2393
Magnesium sulfate Sigma Aldrich M2643
Ammonium chloride Sigma Aldrich A9434
Tris-base
Dimethyl Sulfoxide Sigma Aldrich  D8418
(5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) Molecular Probes C-1157 Reconstitute in DMSO
Phorbol 12,13-dibutyrate (PBDU, Phorbol ester) Sigma Aldrich P1269
A23187 (Calcium ionophore) Sigma Aldrich C7522
Name Company Catalog Number Comments
Antibodies and recombinant protein
CD11b biotin (clone m1/70) Biolegend 101204 T cell depletion cocktail
CD11c biotin (clone N418) Biolegend 117304 T cell depletion cocktail
Gr-1 biotin (clone RB6-8C5) Biolegend 108404 T cell depletion cocktail
Ter119 biotin (clone Ter119) Biolegend 116204 T cell depletion cocktail
TCR-γδ biotin (clone GL-3) Biolegend 118103 T cell depletion cocktail
CD19 biotin (clone 6D5) Biolegend 115504 T cell depletion cocktail
B220 biotin (clone RA3-6B2) Biolegend 103204 T cell depletion cocktail
CD49b biotin (clone DX5) Biolegend 108904 T cell depletion cocktail
CD4 biotin (clone GK1.5) Biolegend 100404 T cell depletion cocktail
CD8 biotin (clone 53-6.7) Biolegend 100704 T cell depletion cocktail
F(ab’)2 goat anti-mouse IgM (plate coated) Jackson ImmunoResearch  115-006-075 50 µl/well for coating (96-well)
Anti-mouse CD40 mAb (plate coated) Pharmingen  553722 50 µl/well for coating (96-well)
Recombinant IL-4 ProSpec  Cyt-282
LPS from E. coli Serotype 055:B5 Sigma Aldrich L-4005
Anti-CD3 (clone clone OKT3) (plate coated) eBioscience  16-0037-85 50 µl/well for coating (96-well)
Anti-CD28 (clone clone 37.51 ) (plate coated)  eBioscience  16-0281-85 50 µl/well for coating (96-well)
Recombinant IL-2 ProSpec Cyt-370
Albumin from chicken egg white, Ovalbumin Sigma Aldrich A7641

Referenzen

  1. Nikolich-Žugich, J., Slifka, M. K., Messaoudi, I. The many important facets of T-cell repertoire diversity. Nat. Rev. Immunol. 4 (2), 123-132 (2004).
  2. LeBien, T. W., Tedder, T. F. B lymphocytes: how they develop and function. Blood. 112 (5), 1570-1580 (2008).
  3. Brownlie, R., Zamoyska, R. T cell receptor signaling networks: branched, diversified and bound. Nat. Rev. Immunol. 13 (4), 257-269 (2013).
  4. Neo, W. H., Lim, J. F., Grumont, R., Gerondakis, S., Su, I. C-rel regulates ezh2 expression in activated lymphocytes and malignant lymphoid cells. J. Biol. Chem. 289 (46), 31693-31707 (2014).
  5. Gunawan, M., et al. The methyltransferase Ezh2 controls cell adhesion and migration through direct methylation of the extranuclear regulatory protein talin. Nat Immunol. 16 (5), 505-516 (2015).
  6. Lyons, A. B., Parish, C. R. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods. 171 (1), 131-137 (1994).
  7. Cabatingan, M. S., Schmidt, M. R., Sen, R., Woodland, R. T. Naïve B lymphocytes undergo homeostatic proliferation in response to B cell deficit. J. Immunol. 169 (12), 6795-6805 (2002).
  8. Bedoya, S. K., Wilson, T. D., Collins, E. L., Lau, K., Larkin, J. Isolation and Th17 differentiation of naïve CD4 lymphocytes. J. Vis. Exp. (79), e50765 (2013).
  9. Su, I., et al. Ezh2 controls B cell development through histone h3 methylation and Igh rearrangement. Nat. Immunol. 4 (2), 124-131 (2003).
  10. Mecklenbräuker, I., Saijo, K., Zheng, N., Leitges, M., Tarakhovsky, A. Protein kinase Cδ controls self-antigen-induced B-cell tolerance. Nature. 416 (6883), 860-865 (2002).
  11. Rush, J. S., Hodgkin, P. D. B cells activated via CD40 and IL-4 undergo a division burst but require continued stimulation to maintain division, survival and differentiation. Eur. J. Immunol. 31 (4), 1150-1159 (2001).
  12. Quah, B. J. C., Warren, H. S., Parish, C. R. Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Nat. Protoc. 2 (9), 2049-2056 (2007).
  13. Quah, B. J. C., Parish, C. R. New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes. J. Immunol. Methods. 379 (1-2), 1-14 (2012).
  14. Hawkins, E. D., Hommel, M., Turner, M. L., Battye, F. L., Markham, J. F., Hodgkin, P. D. Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data. Nat. Protoc. 2 (9), 2057-2067 (2007).
  15. Tomlinson, M. J., Tomlinson, S., Yang, X. B., Kirkham, J. Cell separation: Terminology and practical considerations. J. Tissue Eng. 4 (1), 1-14 (2013).

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Lim, J. F., Berger, H., Su, I. Isolation and Activation of Murine Lymphocytes. J. Vis. Exp. (116), e54596, doi:10.3791/54596 (2016).

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