Materials and Reagents

Pipettes tips (Eppendorf, catalog numbers: 022491105 , 022491113 , 022491121 , 022491164 )
Microcentrifuge tube (Fisher Scientific, catalog number: 05-408-130 )
12-well cell culture plate (Greiner Bio One International, catalog number: 665102 )
40 μm strainer (Corning, Falcon^® , catalog number: 352340 )
Microcentrifuge tube magnet (New England Biolabs, catalog number: S1509S )
Dynabeads: Sheep anti-rat IgG (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 11035 )
LYVE-1 antibody, 0.5 mg/ml (R&D Systems, catalog number: AF2125 )
Trizol solution (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 15596018 )
RNA Prep Kit (QIAGEN, catalog number: 74106 )
Bovine serum albumin (Rockland Immunochemicals, catalog number: BSA-50 )
PBS w/o Ca²⁺ and Mg²⁺ (Merck, Omnipur^®, catalog number: 6505-OP )
Collagenase (Sigma-Aldrich, catalog number: 10103578001 )
Dispase (Sigma-Aldrich, catalog number: D4693-1G )
DNase (Thermo Fisher Scientific, Ambion^TM, catalog number: AM2224 )
HBSS (Thermo Fisher Scientific, Gibco^TM, catalog number: 14175095 )
Ammonium Chloride (NH₄Cl) (Merck, Calbiochem, catalog number: 168320 )
Sodium Bicarbonate (NaHCO₃) (Merck, catalog number: SX0320-1 )
Na₂EDTA (EDTA disodium) (VWR, catalog number: 97061-022 )
Molecular grade water (Sigma-Aldrich, catalog number: W4502-1L )
PBS/BSA Solution (see Recipes)
Enzyme Solution (see Recipes)
RBC Lysis Buffer Solution (see Recipes)

Equipment

Incubator (Fisher Scientific)
Centrifuge (Eppendorf, model: 5415 R )
NanoDrop^TM 2000 Spectrophotometers (Thermo Fisher Scientific, model: NanoDrop^TM 2000 )

Procedure

Dynabead Preparation
1. Pipette 500 μl of Dynabead solution (50 μl/embryo) into a microcentrifuge tube.
2. Place on microcentrifuge tube magnet and remove the solution.
3. Wash the Dynabeads with 1 ml PBS/0.1% BSA for 3 min on gentle rotation.
4. Place the tube on microcentrifuge tube magnet and discard supernatant.
5. Repeat Steps A3-A4 once.
6. Add 1 ml PBS/0.1% BSA.
7. Add 100 μg LYVE-1 mAb (10 μg of antibody/embryo) to the resuspended Dynabead solution.
8. Incubate for 30-45 min at 4 °C with gentle rotation.
9. Place the tube on magnet and discard supernatant.
10. Resuspend Dynabeads by gently pipetting (100 μl/embryo) in 1 ml of PBS/BSA solution. Store at 4 °C until needed.
Embryo Dissection and Endothelial Cell Isolation

Euthanize pregnant mouse at E14.5; Remove uterus and isolate embryos.
Expose the abdomen of the embryo and remove as much liver tissue as possible (see Notes).
Cut embryo into 4-5 smaller pieces and digest tissue in 1 ml of enzyme solution for 30 min at 37 °C. Pipette mixture up and down several times every 10 min.
Using a sterile 12-well cell culture plate and a 40 μm strainer, filter each mixture to remove debris; flush with 0.5-1 ml cold HBSS.
Transfer each filtered solution to a separate microcentrifuge tube.
Spin down cell solution at 600 x g for 1 min at 4 °C. Discard supernatant.
Resuspend in 500 μl RBC lysis buffer, pipet up and down several times. Let rest on countertop for 5 min.
Spin down cells at 600 x g for 1 min at 4 °C.
Resuspend cells in 1 ml cold PBS/0.1% BSA.
Add 100 μl prepared Dynabead-LYVE-1 solution (from Step A10) to each sample.
Incubate for 45 min-1 h at 4 °C with gentle rotation.
Place each tube on magnet for 5 min; discard supernatant.
Wash pellet twice with 1 ml cold PBS/BSA by gentle pipetting up and down several times using a 1 ml pipette tip.
Place each tube on magnet for 5 min; discard supernatant.
Add 0.5 ml Trizol solution to each tube. Use immediately or freeze at -80 °C for later use.
Isolate RNA using QIAGEN prep kit.

Data analysis

After isolating RNA using the isolation kit, RNA purity and quality should be checked prior to running any downstream experiments. For the purpose of this experiment, only samples with a 260/280 ratio of 1.9-2.1 were selected for RT-qPCR experiments. Purity was assessed using a Thermo Fisher Scientific Nanodrop 2000 device. However, standard spectrophotometers can also be used to assess quality.

Notes

The physiological role of the LYVE-1 receptor is still under debate, as well as exactly what tissues express the receptor at which time-point during mouse embryo development. LYVE-1 is highly expressed by sinusoidal liver cells (a specialized type of blood vessel) so removal of the liver prior to digesting the embryo would improve the purity of isolating lymphatic endothelial cells.
While this protocol describes a method for isolating cells and directly obtaining RNA for quantitative real-time PCR analysis, it can be adapted for a variety of downstream purposes. For example, after step 14 of Part B, cells could be eluted from the Dynabeads (according to manufacturer’s instructions), which would allow for alternative downstream methods to be employed (cell culture or fluorescence-activated cell sorting [FACS]) depending on the needs of the individual.

Recipes

PBS/BSA Solution
Add 0.1% bovine serum albumin to PBS w/o Ca²⁺ and Mg²⁺
Enzyme Solution
2.5 mg/ml collagenase
2.5 mg/ml dispase
25 μg/ml DNase
10 ml HBSS
3.RBC Lysis Buffer Solution
150 mM NH₄Cl (Ammonium Chloride) MW = 53.49
2 g
10 mM NaHCO₃ (Sodium Bicarbinate) MW = 84.01
0.21 g
1 mM Na₂EDTA (EDTA disodium) MW = 372.2
0.09 g
Distilled H₂O
250 ml

Acknowledgments

This procedure was developed in the laboratory of Dr. Courtney Griffin at the Oklahoma Medical Research Foundation in Oklahoma City, OK. There are no conflicts of interest.

References

Banerji, S., Ni, J., Wang, S. X., Clasper, S., Su, J., Tammi, R., Jones, M. and Jackson, D. G. (1999). LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J Cell Biol 144(4): 789-801.
Jackson, D. G. (2003). The lymphatics revisited: new perspectives from the hyaluronan receptor LYVE-1. Trends Cardiovasc Med 13(1): 1-7.