Sep 25, 2023
Isolation of mouse brain pericytes (PDGFR-B+) using magnetic sorting (MACS)
- 1Université Laval
Protocol Citation: Daniel Manrique-Castano 2023. Isolation of mouse brain pericytes (PDGFR-B+) using magnetic sorting (MACS). protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmk5bbg3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 01, 2021
Last Modified: September 25, 2023
Protocol Integer ID: 48784
Keywords: Flow Cytometry, Magnetic separation, MACS
Abstract
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:
Guidelines
This protocol makes use of the reagents contained in the Adult Brain Dissociation Kit, mouse and rat (Miltenyi biotec, cat# 130-107-677).
Materials
This protocol makes use of the reagents contained in the Adult Brain Dissociation Kit, mouse and rat (Miltenyi biotec, cat# 130-107-677).
Protocol materials
PBS - Phosphate-Buffered Saline (10X) pH 7.4Thermo Fisher ScientificCatalog #AM9625
Step 2
Debris Removal SolutionMiltenyi BiotecCatalog #130-109-398
Step 10
Red Blood Cell Lysis Solution (10×)Miltenyi BiotecCatalog #130-094-183
In 2 steps
BD Pharmingen™ Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™)BD BiosciencesCatalog #553142
Step 18
LIVE/DEAD™ Fixable BlueThermo Fisher ScientificCatalog #L23105
In 2 steps
UltraComp eBeadsThermo Fisher ScientificCatalog #01-2222-42
Step 20
CD140b AntibodyMiltenyi BiotecCatalog #130-123-271
Step 19
Before start
Read the full protocol
Mouse euthanasia and tissue dissection
Mouse euthanasia and tissue dissection
40m
40m
Euthanize mice employing CO2 or decapitation according to institutional guidelines.
Remove the brain following standard procedures, avoiding tissue damage. Place the brain in falcon tubes or 10-cm Petri dishes filled with 1x PBS - Phosphate-Buffered Saline (10X) pH 7.4Thermo Fisher ScientificCatalog #AM9625 On ice .
Prepare the enzyme mix 1 and 2 according to the table (quantities per brain)
| A | B | C | D | |
| Mix 1 | Mix 2 | |||
| Enzyme P | Buffer Z | Buffer Y | Enzyme A | |
| 50 ul | 1900 ul | 20 ul | 10 ul | |
Using gentleMAC Octo Dissociator27 steps
Device: Cat# 130-096-427
Tubes: Cat # 130-093-237
Transfer the brains to a 10-cm Petri dish. Use forceps and a razor blade/surgical scalpel to mince the tissue into small pieces (approx 1 mm x 1mm).
Place the pieces into the tubes filled with Mix1 and Mix2. Close the cap tightly to prevent liquid leakage.
Mouse euthanasia and tissue dissection
Mouse euthanasia and tissue dissection
40m
40m
Place the C tubes into the octo dissociator with heaters and execute the program 37C_ABDK_1
Cell dissociation and enzymatic digestion
Cell dissociation and enzymatic digestion
35m
35m
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.
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.
Divide the sample in 2 15 mL falcon tubes.
Note
This step favors the coming debris removal. In our experience, the division of a single hemisphere into two samples gives optimal results.
Centrifugate the samples at 300 x g, 4°C, 00:05:00
5m
Debris removal
Debris removal
15m
15m
Decant the supernadant and resuspend the pellet in 3100 µL of D-PBS + 900 µL Debris Removal SolutionMiltenyi BiotecCatalog #130-109-398 .
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. .
Note
We recommend doing this manually, controlling the PBS fluid with the thumb
Centrifuge 3000 x g, 4°C, 00:10:00 with reduced acceleration and brake
Acceleration: 1
Brake: 1
Note
If the acceleration and brake are at full speed, the debris removal is sub-optimal.
10m
Aspirate the first two phases and fill up the tube up to 5 mL with PBS. Gently invert the tube three times.
Centrifugate 1000 x g, 4°C, 00:10:00 and decant supernadant.
10m
Red Blood Cell Removal (Optional step)
Red Blood Cell Removal (Optional step)
15m
15m
Dilute the Red Blood Cell Lysis Solution (10×)Miltenyi BiotecCatalog #130-094-183 at 1:10 using double-distilled water (ddH2O).
Note
IMPORTANT: Use only ddH2O, not distilled water or other buffers.
Resuspend the pellet in 1 mL of Red Blood Cells Removal Solution, transfer to FACS tubes, and incubate for 00:10:00 in the refrigerator 4 °C
Note
If the samples have been divided before debris removal, they can be merged again in this step. Each sample can be homogenized in 500 µL of Red Blood Cell Lysis Solution (10×)Miltenyi BiotecCatalog #130-094-183 and then merged in a single FACS tube.
10m
When incubation is finished, add 4 mL of cold HBSS/BSA/Glucose buffer, and centrifuge 300 x g, 4°C, 00:05:00 . Aspirate the supernatant afterwards.
5m
Cell staining
Cell staining
35m
35m
Resuspend the cells in 100 µL of HBSS/BSA/Glucose buffer and add 1 µL of BD Pharmingen™ Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™)BD BiosciencesCatalog #553142
. Incubate for 10 minutes at 4 °C .
Add 10 µL of CD140b AntibodyMiltenyi BiotecCatalog #130-123-271 (1:10 dilution) and 1 µL of LIVE/DEAD™ Fixable BlueThermo Fisher ScientificCatalog #L23105 (1:100 dilution) to label dead cells.
Incubate for 00:30:00 at 4 °C in the dark.
Note
These steps involving control sample preparation are only suitable if subsequent FACS is intended. For MACS (only) experiments, incubation of the experimental samples with the blocking solution and desired antibody is sufficient. Go directly to step 22.
If additional stainings are performed, add the required antibody amount according to titration and set corresponding controls = Single-color positive controls and fluorescence minus one (FMO) controls.
.
30m
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 UltraComp eBeadsThermo Fisher ScientificCatalog #01-2222-42 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 LIVE/DEAD™ Fixable BlueThermo Fisher ScientificCatalog #L23105 (1:100 dilution).
Incubate the third control sample (together with the experimental samples) for 00:30:00 .
30m
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, 4°C, 00:05:00
5m
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).
Add 20 µL of magnetic beats according to your antibody incubation (Anti-PE magnetic beats or anti-APC magnetic beats) and incubate for 00:15:00 at 4 °C .
15m
Prepare and label FACS or Eppendorf tubes to collect the negative fraction (140b negative cells), and positive fractions (140b positive cells).
When incubation with the magnetic beats is finished, add 2 mL of HBSS/BSA/Glucose buffer and 300 x g, 4°C, 00:10:00 . Discard the supernatant, and resuspend the cells in 500 µL of PBS + 2% FCS
Note
PBS is recommended at this step to avoid excessive bubbling during MACS.
10m
Magnetic Separation (MACS sorting)
Magnetic Separation (MACS sorting)
5m
5m
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.
Note
Place a container below the magnet to collect buffer remains.
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.
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).
Note
To increase the purity of 140b positive cells, the previous step can be repeated using a new magnetic collum.
Centrifugate the samples 300 x g, 4°C, 00:05:00 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.
Note
Depending on the experimental porpuses, the experimenter can discard the negative fractions collected during magnetic separation.
Consider that the positive fraction gives strong interference bands at the level of 60 Kda in western blots given the presence of beats.
5m