#9530 DRAFT
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Customer protocol
Determination of parasite load from skin
tissue infected with Leishmania major
Beate Lorenz and Esther von Stebut-Borschitz*
Department of Dermatology, Johannes-Gutenberg
University, Mainz, Germany
* Corresponding author ([email protected])
Methods
1.Prepare collagenase medium freshly.
2.Split mouse ear into to halves along the cartilage.
3.Add 1.5 mL collagenase medium per 6-well and
incubate one mouse ear for 2 hours at 37 °C, 5% CO₂.
4.Transfer both halves of one ear into a gentleMACS
C Tube.
5.Tightly close the C Tube and attach it upside down
onto the sleeve of the gentleMACS Dissociator.
6.Run the defined gentleMACS Program (refer to
figure 1 and table 1). For more information about
creating new programs, refer to the gentleMACS
Dissociator user manual.
7.After termination of the program, detach C Tube
from the gentleMACS Dissociator.
8.Discard cap of C Tube and replace it by a cap of a
gentleMACS M Tube and run gentleMACS Program B.
9.Apply cell suspension to a cell strainer (70 µm mesh
size) placed on a 50 mL tube.
10.Wash the cell strainer twice with 5 mL PBS.
11.Discard cell strainer and centrifuge sample at
3000×g for 8 minutes.
12.Resuspend cell pellet in 1 mL drosophila medium.
13.Serially dilute 100 µL sample to determine parasite
load.
Background
In cutaneous Leishmania major (L. major) infections of mice,
the existence of Th1/Th2 cells has been identified more
than 20 years ago. Nowadays, the role of additional T cell
populations as well as B cell–mediated immunity are well
established. Granuloma formation in mice is dependent on
the ability to efficiently mount anti-Leishmania immunity.
If this process fails, early parasite dissemination into, e.g.,
visceral organs, occurs.
This protocol describes a model for determining lesional
parasite loads after experimental L. major infection for
a correlation to lesion sizes at the inoculation site.
Both the size of the lesion as well as information about
parasite containment versus spreading to visceral organs
are important parameters for determining disease outcome
in mice.
Materials and methods
Materials
• gentleMACS Dissociator™ or
gentleMACS Octo Dissociator
• gentleMACS C Tubes and M Tubes
• Incubator (37 °C, 5% CO₂)
• 6-well cell culture plate
• Cell strainer (70 µm mesh size)
• Collagenase medium (100 mg collagenase and 1 mL
Penicillin and Streptomycin per 100 mL DMEM medium)
• Drosophila medium
• Phosphate-buffered saline (PBS)
4000
3000
rpm
2000
1000
0
10
20
30
-1000
-2000
Time in seconds
Figure 1: User-defined gentleMACS Program.
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The content of this publication has not been verified by Miltenyi Biotec.
Command
Revolutions
per minute
(rpm)
Time [min]
Time [sec]
spin
700
0
1
spin
–400
0
1
spin
1100
0
2
spin
–600
0
1
spin
1700
0
3
spin
–1200
0
1
spin
2500
0
5
spin
–1400
0
1
spin
3300
0
4
spin
2500
0
5
spin
–1400
0
1
spin
3300
0
4
Conclusion
spin
3700
0
8
spin
0
0
1
Table 1: Defines the time given in seconds (sec) required to reach the
needed speed. This is given in revolutions per minute (rpm).
L. major parasite load analysis can be accomplished
with ease, saving of time, and high reproducibility
using a robust experimental set up based on the
gentleMACS Octo Dissociator.
Results
References
The gentleMACS Octo Dissociator provides identical
high quality results of parasitic loads 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.
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.
gentleMACS Octo Dissociator
gentleMACS Octo Dissociator
5 minutes
Figure 3: 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.
Competitor M
10¹⁰
Parasites/ear
Competitor M
57 minutes
10⁸
10⁶
10⁴
10²
Week 3
BALB/c
Week 3
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Week 6
C57BL/6
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The content of this publication has not been verified by Miltenyi Biotec.
V. 02
Figure 2: BALB/c and C57BL/6 mice were infected with L. major (2×10⁵
parasites per ear). Parasite load was determined 3 and 6 weeks later.