TITLE: Isolation of ECs from Brain tissue for scRNAseq on the 10x Chromium
AUTHORS: Guido  Krähenbühl

[DESCRIPTION]
Protocols for endothelial cell isolation from mouse tissues: brain, addapted from  

Open Access • DOI: 10.1016/j.xpro.2021.100508

[STEPS]
SECTION: Preparation
1. Day before experiment check Antibodies, Enzyme Mix 1 and 2 Reagents to be used and prepare the following Buffer:

 

 

 

 
SECTION: Preparation
2. Before starting prepare:
Dissection Tools
20G needle and thin syringe
If Brain tissue is also taken, prepare perfusion kit
10mL DMEM Genta

 


Keep all reagents on ice
SECTION: Tissue Collection
3. Sacrifice mice using Isofluorine 
(do not use cervical dislocation as this might affect perfusion of the brain)
SECTION: Tissue Collection
4. Make an incision in the skin with fine scissors about a couple of millimeters above the orifice. From this opening the incision must continue on the mid ventral side up to the chin.

SECTION: Tissue Collection
5. Perform transcardial perfusion via the left ventricle with 8 mL  / mouse
 at a perfusion rate of 2 mL/min for 5 min.
SECTION: Tissue Collection
6. If cervical lymph nodes are needed disect them now, otherwise remove the Head and harvest Brain regions of interest and collect all Brains in 10 mL 

SECTION: Tissue Collection
7. If lymph nodes are also analysed    and repeat for up to 5 mice.
If only brain ECs are analysed a total of 2 mice should suffice

SECTION: Tissue processing
8. Mince Brain samples with a scalpel in a 10cm dish until they may pass through a cut 1000P pipette
SECTION: Tissue processing
9. Transfer minced sample to a 15 mL conical tube and add 1950 µL  at    on the tube rotator

SECTION: Tissue processing
10. Add 30 µL 
SECTION: Tissue processing
11.  Pipette 10 times up and down with a pasteur pipette (until the suspension passes without clogging)
SECTION: Tissue processing
12.  Incubate at   on the tube rotator
SECTION: Tissue processing
13. Pass the cell supsension 10 times through a 20G needle on a 1mL syringe (dont create bubbles or foam)
SECTION: Tissue processing
14.  Incubate at    on the tube rotator
SECTION: Tissue processing
15. Transfer cell suspension to a 50 mL conical tube, add 40 mL HBSS and filter through a 70 um cell strainer.
SECTION: Tissue processing
17. Discard Supernatant and pool 3 pellets in 3600 µL per Brain, add 400 µL per Brain and incubate at 4 °C  for 15 min 

SECTION: Tissue processing
16. Centrifuge at 300 x g, 5 min, 4 °C 

SECTION: Tissue processing
16.1. Transfer supernatant to new 50 mL conical tube, keep pellet on ice and centrifuge supernatant at 300 x g, 5 min, 4 °C 
SECTION: Tissue processing
16.2. Transfer supernatant to new 50 mL conical tube, keep pellet on ice and centrifuge supernatant at 300 x g, 5 min, 4 °C 
SECTION: Tissue processing
18. Add a total volume of 40 mL  and centrifuge at 300 x g, 5 min, 4 °C 
Prepare 6 times the number of LS columns per Brain by rinsing each with 3mL MACS buffer
SECTION: Tissue processing
19. Resuspend in 6 mL per Brain sample and run 1mL per LS column (collect elluent)

SECTION: Tissue processing
19.1. Wash each column twice with 1mL MACS buffer (collect elluent)
SECTION: Tissue processing
20. Centrifuge total eluent at 300 x g, 5 min, 4 °C , remove supernatant

SECTION: Tissue processing
21. Pool Pellets in 2 mL per Brain and centrifuge at 300 x g, 5 min, 4 °C 

SECTION: FACS sorting
22. Resuspend the pellet in 500 µL per brain

SECTION: FACS sorting
23. Prepare Controls and Samples with each antibody at 1/200:
Cells only
CD31
CD45
Viability dye
PDPN

SECTION: FACS sorting
24. Stain Cells for 30 min at 4 °C 

SECTION: FACS sorting
25. Add 3mL of FACS Buffer, centrifuge at 300 x g, 5 min, 4 °C and resuspend in 2 mL of FACS Buffer