#9530 DRAFT
Sharing ideas, sharing success
For users, by users
Customer protocol
Isolation of lymphatic endothelial cells (LECs) from
mouse embryos using the gentleMACS™ Dissociator
Materials and methods
Lara Planas-Paz* and Eckhard Lammert
Institute of Metabolic Physiology, Heinrich-Heine University,
Düsseldorf, Germany
* Corresponding author ([email protected])
Materials
• gentleMACS Dissociator or
gentleMACS Octo Dissociator
• gentleMACS C Tubes
• Red Blood Cell Lysis Solution (10×)
• Hank’s Balanced Salt Solution (HBSS) with Ca²+ and Mg²+
• Collagenase II solution 10.000 U/mL
• DNase I solution 30.000 U/mL
• PEB buffer: Prepare a solution containing
­phosphate-buffered saline (PBS), pH 7.2,
0.5% bovine serum albumin (BSA),
and 2 mM EDTA. Degas buffer before use.
• Anti-FITC MultiSort Kit
• Rat anti-mouse PECAM-1 FITC- conjugated monoclonal
antibody (clone: 390)
• Rabbit anti-mouse Lyve-1 antibody
• peqGOLD TriFast™
Background
The lymphatic system exerts vital functions in vertebrate
tissues during development, health and disease. Lymphatic
vessels remove interstitial fluid that accumulates within the
tissues due to transvascular fluid passage, and transport it
back to the blood vasculature thus closing the fluid cycle
in the living organism. When the interstitial fluid pressure
rises, lymphatic endothelial cells (LECs) are stretched due to
changes in the surrounding extracellular matrix (ECM) that
they adhere to, and their intercellular junctions are opened
to absorb interstitial fluid. According to the current model,
stretching of LECs in response to an increased interstitial
fluid pressure is key to the removal of extracellular fluid
from vertebrate tissues and to the expansion of the
lymphatic vasculature. Lymphatic vessels also play an
important role in pathologies such as during inflammation,
cancer cell dissemination, lymph node metastasis, and
lymph edema.
Lymphatic vessels are formed by LECs, which differ from
blood vascular endothelial cells by the expression of a
number of molecular markers, including Lyve-1, Prox1 and
vascular endothelial growth factor-3 receptor (VEGFR3).
We designed experiments¹ to identify a correlation between
the amount of interstitial fluid, the stretching of LECs, and
the extent of lymph vessel expansion in the developing
mouse embryo. By using both loss-of-fluid and gain‑of‑fluid
experiments, we showed that the fluid volume within the
interstitium of embryonic and adult mice controls LEC
elongation, VEGFR3 signaling and LEC proliferation in a β1
integrin- and VEGF-C–dependent manner.
This protocol describes the procedure to isolate LECs from
mouse embryos using the gentleMACS™ Dissociator.
Methods
1. Isolate mouse embryos (embryonic day (E) 12.0 was
the earliest stage used) and rinse in cold HBSS.
2. Transfer the upper half of each embryo into a
gentleMACS C Tube containing 4.7 mL HBSS.
3. Add 300 µL Collagenase II solution (600 U/mL)
and 10 µL DNase I solution (60 U/mL).
4. Tightly close the C Tube and attach it upside down
onto the sleeve of the gentleMACS Dissociator.
5. Run gentleMACS Program m_heart_01.
6. Incubate for 30 minutes at 37 °C and turn every
5–10 minutes to resuspend settled tissue fragments.
7. Attach C Tube upside down onto the sleeve of the
gentleMACS Dissociator.
8. Run gentleMACS Program m_heart_02.
9. Spin down the tube. Resuspend the sample and apply
the cell suspension to a 70 µm cell strainer placed onto
a 50 mL tube.
1/2
The content of this publication has not been verified by Miltenyi Biotec.
10. Wash the cell strainer with 5 mL cold HBSS
and centrifuge at 300×g for 10 minutes.
11. Aspirate the supernatant completely and apply 5–10 mL
1× Red Blood Cell Lysis Solution.
12. Incubate for maximal 2 minutes at room temperature
(RT). Centrifuge at 300×g for 10 minutes and aspirate
the supernatant completely.
13. Wash the cell suspension with PEB buffer.
14. Label single-cell suspensions with anti-mouse PECAM-1
and anti-mouse Lyve-1 antibodies to sort LECs in a
multi-step manner. Follow the protocol in the data
sheet of the Anti-FITC MultiSort Kit.
15. The sorted LECs are finally resuspended in 500–1000 µL
peqGOLD TriFast and RNA is extracted.
A
*
β1 integrin mRNA expression
in sorted LECs (% of control)
140
120
100
80
60
40
20
0
β1 int ∆/+
β1 int ∆/∆
LECs of E12.0–E12.5
B
LECs of
β1 int ∆/+
Results
LECs of
β1 int ∆/∆
WT
embryo
β1 int ∆/∆
WT
–RT
β1 integrin
Adapted from our work, we propose a new signaling
mechanism, which is based on mechanotransduction and
is essential for lymphatic growth and fluid homeostasis in
the mammalian embryo.
β2-microglobulin
Conclusion
β1 integrin mRNA expression
in sorted LECs normalized to
β2-microglobulin (A.U.)
Isolation of LECs from mouse embryos can be accomplished
with ease using the gentleMACS Dissociator.
References
1. P
lanas-Paz, L. et al. (2012) Mechanoinduction of lymph
vessel expansion. EMBO J. 31(4): 788–804.
2. Schwartz, M.A. and Simons, M. (2012) Lymphatics thrive
on stress: mechanical force in lymphatic development.
EMBO J. 31(4): 781–782.
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0
β1 int ∆/+
Figure 1: β1 integrin deletion in LECs sorted by MACS® Technology
from E12.0–E12.5 mouse embryos.
(A) β1 integrin mRNA expression as determined by real-time RT-PCR
of LECs sorted from β1 integrinΔ/+ (grey column) and β1 integrinΔ/Δ
(orange column) E12.0–E12.5 mouse embryos. All values are means ±SD,
n≥5 mouse embryos per genotype, *p = 0.0005.
(B) RT-PCR products for β1 integrin and β2-microglobulin of LECs sorted
from β1 integrinΔ/+ and β1 integrinΔ/Δ E12.0–E12.5 mouse embryos.
Total RNA from an E12.5 wild type embryo was used as positive control.
The lower graph shows β1 integrin mRNA expression normalised to
β2-microglobulin mRNA expression of sorted LECs from β1 integrinΔ/+
(grey columns) and β1 integrinΔ/Δ (orange columns) E12.0–E12.5 mouse
embryos, and of an E12.5 wild type embryo (yellow column).
A.U.: arbitrary units.
Reproduced with kind permission from the NPG and EMBO J.
Miltenyi Biotec provides products and services worldwide. Visit www.miltenyibiotec.com/local to find your nearest Miltenyi Biotec contact.
Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.
gentleMACS and MACS are trademarks or registered trademarks of Miltenyi Biotec GmbH. All other trademarks mentioned in this document are the property of
their respective owners and are used for identification only. Copyright © 2012 Miltenyi Biotec GmbH. All rights reserved.
2/2
The content of this publication has not been verified by Miltenyi Biotec.
V. 01
Visit www.gentleMACS.com for more
information on Miltenyi Biotec's sample preparation
portfolio or find more customer protocols on
www.gentleMACS.com/protocols