TITLE: Isolation of mouse brain pericytes (PDGFR-B+) using magnetic sorting (MACS)
AUTHORS: Daniel Manrique-Castano

[DESCRIPTION]
This protocol employs a modified procedure of the Adult Brain Dissociation kit (Miltenyi Biotec) to sort PDGFR-B+ cells from mouse brains. The protocol is adapted to enhance cell recovery and survival. The original protocol can be seen in:

https://www.miltenyibiotec.com/CA-en/products/adult-brain-dissociation-kit-mouse-and-rat.html#130-107-677

[BEFORE_START]
Read the full protocol

[GUIDELINES]
This protocol makes use of the reagents contained in the Adult Brain Dissociation Kit, mouse and rat (Miltenyi biotec, cat# 130-107-677).

[STEPS]
SECTION: Mouse euthanasia and tissue dissection
1. Euthanize mice employing CO2 or decapitation according to institutional guidelines.
SECTION: Mouse euthanasia and tissue dissection
2. Remove the brain following standard procedures, avoiding tissue damage. Place the brain in falcon tubes or 10-cm Petri dishes filled with 1x   on ice  .
4. Place the pieces into the tubes filled with Mix1 and Mix2. Close the cap tightly to prevent liquid leakage.


SECTION: Mouse euthanasia and tissue dissection
3. Prepare the enzyme mix 1 and 2 according to the table (quantities per brain)

 
 
SECTION: Cell dissociation and enzymatic digestion
6. When the digestion is complete, resuspend the samples and add them into a moisten 70 µL cell filter attached to a 50 mL falcon tube. Do not discard 15 mL tubes at this point.

SECTION: Cell dissociation and enzymatic digestion
7. Add 7 mL of cold D-PBS to the falcon tubes used for digestion. Mix briefly to recover any sample left. Pass the buffer through the filter using a glass pipette. Discard the filter and the 15 mL falcon tubes.

SECTION: Cell dissociation and enzymatic digestion
9. Centrifugate the samples at 300 x g, 5 min, 4 °C 

SECTION: Debris removal
10. Decant the supernadant and resuspend the pellet in 3100 µL of D-PBS + 900 µL   .

SECTION: Debris removal
11. In this step, a gradient is performed. Overlay very gently 4 mL of cold D-PBS using a glass pipet. In the end, two phases must be easily recognizable. .
 


SECTION: Debris removal
12. Centrifuge 3000 x g, 10 min, 4 °C with reduced acceleration and brake

Acceleration: 1
Brake: 1
 

SECTION: Debris removal
13. Aspirate the first two phases and fill up the tube up to 5 mL with PBS. Gently invert the tube three times.  
SECTION: Debris removal
14. Centrifugate  1000 x g, 10 min, 4 °C and decant supernadant.
SECTION: Red Blood Cell Removal (Optional step)
15. Dilute the   at 1:10 using double-distilled water (ddH2O).
 

SECTION: Red Blood Cell Removal (Optional step)
16. Resuspend the pellet in 1 mL of Red Blood Cells Removal Solution, transfer to FACS tubes, and incubate for 10 min in the refrigerator 4 °C
 


SECTION: Red Blood Cell Removal (Optional step)
17. When incubation is finished, add 4 mL of cold HBSS/BSA/Glucose buffer, and centrifuge 300 x g, 5 min, 4 °C . Aspirate the supernatant afterwards.  

SECTION: Cell staining
18. Resuspend the cells in 100 µL of HBSS/BSA/Glucose buffer and add 1 µL of   
. Incubate for 10 minutes at 4 °C .

SECTION: Cell staining
19. Add 10 µL of  (1:10 dilution) and 1 µL of   (1:100 dilution) to label dead cells. 
Incubate for 30 min at 4 °C in the dark.
 


.
SECTION: Cell staining
20. At this point, the researcher should also set the staining controls for the experiment. For this simple setup, prepare:

1) A sample containing 100-200 µL  of unstained cells ( Control is ready for FACS)
2) A FACS tube containing 1 drop of  1 µL of antibody, and 100 µL of HBSS/BSA/Glucose buffer (Control readyACS)  
3)  A FACS tube containing one drop of ArC reactive beads (green cap), 1 µL of     (1:100 dilution). 


SECTION: Cell staining
22. When antibody incubation is finished, add 2 mL of HBSS/BSA/Glucose buffer to the samples and control number 3 and centrifuge 300 x g, 5 min, 4 °C

SECTION: Cell staining
23. Decant the supernatant. In control sample 3, add 1 drop of ArC negative beads (white cap) and 100 µL of HBSS/BSA/Glucose buffer. This control sample is ready for FACS. 

Otherwise, experimental samples must be resuspended in 80 µL of HBSS/BSA/Glucose buffer  to perform magnetic separation (MACS sorting).
SECTION: Magnetic Separation (MACS sorting)
27. Place the magnetic columns in a suitable MACS separator and rinse each column (MS columns) with 500 µL  of PBS + 2% FCS until the buffer is decanted entirely by gravity.
 


SECTION: Magnetic Separation (MACS sorting)
28. Using a FACS tube rack, match negative fractions FACS (or Eppendorf) tubes to each column and apply the 500 µL of cell suspension into each column. When the buffer reservoir is empty, add 500 µL of PBS + 2% FCS three times.

25. Prepare and label FACS or Eppendorf tubes to collect the negative fraction (140b negative cells), and positive fractions (140b positive cells).
SECTION: Magnetic Separation (MACS sorting)
29. When all the negative fraction is collected, remove the columns from the magnet and place it on FACS or Eppendorf tubes intended to collect the 140b positive cells. Pipette 1 mL   of PBS + 2% FCS into the columns and firmly push the plunger into the column.

In the end, the research has a tube with a negative fraction (all cells not PDGFR-B+) and a positive fraction (PDGFR-B+ labeled with magnetic beats).
 


SECTION: Magnetic Separation (MACS sorting)
30. Centrifugate the samples 300 x g, 5 min, 4 °C and resuspend in 300 µL of PBS + 2% FCS. Samples are ready for FACS. 

If FACS is not performed, aspirate the supernatant and freeze the cells, or place Othem in an appropriate buffer for subsequent experimental procedures. 
 


SECTION: Cell staining
24. Add 20 µL of magnetic beats according to your antibody incubation (Anti-PE magnetic beats or anti-APC magnetic beats) and incubate for 15 min at 4 °C .

26. When incubation with the magnetic beats is finished, add 2 mL of HBSS/BSA/Glucose buffer and  300 x g, 10 min, 4 °C . Discard the supernatant, and resuspend the cells in 500 µL of PBS + 2% FCS 
 


SECTION: Cell staining
21. Incubate the third control sample (together with the experimental samples) for 30 min .
SECTION: Cell dissociation and enzymatic digestion
8. Divide the sample in 2 15 mL falcon tubes.
 


SECTION: Mouse euthanasia and tissue dissection
4. Transfer the content to 15 mL Falcon tubes and close the cap tightly to prevent liquid leakage.






SECTION: Mouse euthanasia and tissue dissection
5. Place the falcon tubes into a hybridization chamber at 36.5 °C  with constant rotation for 40 min 

We employ the Roto-Therm rotator in the mix mode at 10-speed.

 


SECTION: Mouse euthanasia and tissue dissection
5. Place the C tubes into the octo dissociator with heaters and execute the program 37C_ABDK_1