#9530 DRAFT
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Customer protocol
Isolation of T cells from mouse ear using
the gentleMACS™ Dissociator
Beate Lorenz and Esther von Stebut-Borschitz*
Department of Dermatology, Johannes-Gutenberg
University, Mainz, Germany
* Corresponding author ([email protected])
Methods
1.Prepare Liberase medium freshly.
2.Split ear into to halves along the cartilage.
3.Add 1.5 mL Liberase medium per 6-well
and incubate 1 mouse ear for 1.5 hours
at 37 °C (5% CO₂).
4.Add 1.5 mL RPMI medium per 6-well
for inactivation.
5.Transfer both halves of one ear into a gentleMACS
C Tube containing 1 mL RPMI medium.
6.Tightly close the C Tube and attach it upside down
onto the sleeve of the gentleMACS Dissociator.
7.Run the gentleMACS Program C.
8.Once the program is finished, detach C Tube
from the gentleMACS Dissociator.
9.Apply sample to a cell strainer (70 µm mesh size)
placed on a 50 mL tube.
10.Discard cell strainer and centrifuge sample
at 200×g for 8 minutes.
11.Resuspend pellet in PBS and continue with cell
separation using CD4 and CD8 MicroBeads
according the manufacturers recommendations.
Background
Leishmaniasis is an endemic disease with manifold clinical
manifestations. About 90% of infections caused by different
species of the protozoan parasite Leishmania spp. manifest
as the localized cutaneous form of the disease.
Healing of Leishmania infections is based on dendritic
cell–¹‑³ and T cell–dependent immunity, including
IFN-γ secretion of both CD4+ and CD8+ T cells.⁴‑⁶ Basic
leishmaniasis research is mainly based on mouse models.⁷,⁸
Representative sample preparation after initial intradermal
infection of mouse skin tissue is required for any further
immunological research.
This protocol describes how single-cell suspensions were
obtained from infected mouse ear with the gentleMACS™
Octo Dissociator. Magnetic MicroBeads were used for
reliable isolation of CD4 and CD8 T cells afterwards.
Materials and methods
Results
Materials
• gentleMACS Dissociator or
gentleMACS Octo Dissociator
• gentleMACS C Tubes
• Incubator (37 °C, 5% CO₂)
• Centrifuge
• 6-well cell culture plate
• Cell strainer (70 µm mesh size)
• CD4 (L3T4) MicroBeads, mouse
and CD8a (Ly-2) MicroBeads, mouse
• L iberase™ stock solution with a concentration of 25 mg/mL
• Liberase medium (RPMI medium including 160 µL
Liberase stock solution and 500 µL penicillin/
streptavidin per 10 mL)
• RPMI medium (including 5% fetal bovine serum)
• Phosphate-buffered saline (PBS)
The gentleMACS Octo Dissociator provides more efficient
results of T cell isolation compared to a competitive
instrument used for automated mechanical tissue
disaggregation, which was well established in our lab.
In addition to high quality results the gentleMACS
Octo Dissociator is an easy-to-use system that provides
users with the advantages of increased user safety and
significant reduction of labor time.
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The content of this publication has not been verified by Miltenyi Biotec.
Conclusion
CD4+ T cells ×10⁶
2
Isolation of T cells from mouse ear can be accomplished with
ease and high reproducibly using a robust experimental
setup based on the gentleMACS Octo Dissociator.
References
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1.5
1.Woelbing, F. et al. (2006) Uptake of Leishmania major
by dendritic cells is mediated by Fcγ receptors and
facilitates acquisition of protective immunity. J. Exp.
Med. 203: 177–188.
2.Kautz-Neu, K. et al. (2011) Langerhans cells are important
negative regulators of the anti-Leishmania response. J.
Exp. Med. 208: 885–891.
3.Kautz-Neu, K. et al. (2011) A role for leukocyte-derived
IL-1RA in DC homeostasis revealed by increased
susceptibility of IL-1RA-deficient mice to cutaneous
leishmaniasis. J. Invest. Dermatol. 131: 1650–1659.
4.Kronenberg, K. et al. (2010) Vaccination with TAT-antigen
fusion protein induces protective, CD8+ T cell-mediated
immunity against Leishmania major. J. Invest. Dermatol.
130: 2602–2610.
5.von Stebut, E. and Udey, M.C. (2004) Requirements
for Th1-dependent immunity against infection with
Leishmania major. Microbes. Infect. 6: 1102–1109.
6.Belkaid, Y. et al. (2002) CD8+ T cells are required for
primary immunity in C57BL/6 mice following low-dose,
intradermal challenge with Leishmania major.
J. Immunol. 168: 3992–4000.
7.Scott, P. and Farrell, J.P. (1998) Experimental cutaneous
leishmaniasis: induction and regulation of T cells
following infection of mice with Leishmania major.
Chem. Immunol. 70: 60–80.
8.Lohoff, M. et al. (1998) The Th1/Th2 paradigm
and experimental murine leishmaniasis. Int. Arch.
Allergy Immunol. 115: 191–202.
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gentleMACS™
Octo Dissociator
Competitor M
Figure 1: The amounts of T cells isolated from the infected mouse
ear were significantly higher when isolated with CD4 MicroBeads in
combination with the gentleMACS Octo Dissociator compared to
competitor instrument M. Single-cell suspensions from 5 mice were
pooled and separated by using MicroBeads afterwards.
gentleMACS Octo Dissociator
5 minutes
Competitor M
57 minutes
Figure 2: Comparison of labor time; 8 mouse ears were prepared with
the gentleMACS Octo Dissociator and a competitive instrument in
parallel. Using the gentleMACS Octo Dissociator saves about 1 hour
of working time compared to an instrument frequently used to deliver
automated mechanical tissue disaggregation.
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