PEB buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution (# 130‑091‑376) 1:20 with autoMACS® Rinsing Solution (# 130‑091-222). Keep buffer cold (2−8 °C). Always use freshly prepared buffer. Do not use autoMACS Running Buffer or MACSQuant® Running Buffer as they contain a small amount of sodium azide that could affect the results. 

▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as mouse or rat serum albumin, mouse or rat serum, or FBS. Buffers or media containing Ca²⁺ or Mg²⁺ are not recommended for use.
 

Coat the culture dish with 100 μg/mL fibronectin (overnight).
 

▲ MicroBeads concentrations below are optimized for the processing of 10⁷ total cells from neonatal mouse brain.

▲ For details on the use of the gentleMACS™ Dissociators, refer to the gentleMACS Dissociator user manuals.

1. Dissociate mouse neonatal brain according to the protocol of the Neural Tissue Dissociation Kit (P) including post-dissociation wash steps. 
2. Determine cell number.
3. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.

Download data sheet

Neural Tissue Dissociation Kit (P)
 

▲Volumes for magnetic labeling given below are for up to 1×10⁷ total cells. When working with fewer than 1×10⁷ cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×10⁷ total cells, use twice the volume of all indicated reagent volumes and total volumes).

▲For optimal performance it is important to obtain a single‑cell suspension before magnetic labeling. Pass cells through 70 μm nylon mesh (Pre-Separation Filters (70 μm), # 130-095-823) to remove cell clumps which may clog the column. Moisten filter with buffer before use.

▲Always wait until the column reservoir is empty before proceeding to the next step. 

▲The recommended incubation temperature is 2–8 °C. Higher temperatures and/or longer incubation times may lead to non‑specific cell labeling. Working on ice may require increased incubation times.
 

1. Resuspend cells in 90 μL of PEB buffer per 1×10⁷ total cells.
2. Add 10 μL of CD45 MicroBeads.
3. Mix well and incubate for 15 minutes in the refrigerator (2−8 °C).
4. Wash cells by adding 1 mL of PEB buffer per 1×10⁷ cells and centrifuge at 300×g for 5 minutes. Aspirate supernatant completely.
5. Place LD Column in the magnetic field of a suitable MACS Separator.
   ▲ Note: Automated separation can be performed using the autoMACS Pro Separator with the following program: Depletes.
6. Prepare column by rinsing it with 3 mL of buffer.
7. Apply cell suspension onto the column.
8. Collect unlabeled cells that pass through. Perform three washing steps with 3 mL of PEB buffer each.
9. Collect total effluent; this is the CD45^– fraction.
10. (Optional, if CD45⁺ cells are needed) Remove column from the separator and place it on a suitable collection tube. Pipette 5 mL of PEB buffer onto the column. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column.
11. Proceed with enrichment of CD31⁺ cells.
     

1. Go on with the CD45^– fraction. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
2. Resuspend cell pellet in 90 μL of PEB buffer.
3. Add 10 μL of CD31 MicroBeads.
4. Mix well and incubate for 15 minutes in the refrigerator (2−8 °C).
5. Wash cells by adding 1 mL of PEB buffer and centrifuge at 300×g for 5 minutes. Aspirate supernatant completely.
6. Place MS Column in the magnetic field of a suitable MACS Separator.
   ▲ Note: Automated separation can be performed by using the autoMACS Pro Separator with the following program: Posseld.
7. Prepare column by rinsing with 1 mL of PEB buffer.
8. Apply cell suspension onto the column.
9. Collect unlabeled cells that pass through. Perform three washing steps with 0.5 mL of PEB buffer each.
10. Collect total effluent; this is the CD45^–/CD31^– cell fraction.
11. Remove column from the separator and place it on a suitable collection tube.
12. Pipette 1 mL of PEB buffer onto the column. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column; this is the CD45^–/CD31⁺ target cell fraction.
13. To increase the purity of CD31⁺ cells, the eluted fraction can be enriched over a second MS Column. Repeat the magnetic separation procedure as described in steps 6 to 12 using a new column.
14. Proceed with flow cytometric analysis.
     

▲ For a detailed immunofluorescent staining protocol, refer to the data sheet of CD31 Antibodies, anti-mouse.

1. Incubate cells with CD31 Antibodies, anti-mouse.
2. Analyze cells using a flow cytometer, e.g., the MACSQuant Analyzer 10.

1. Plate 5×10⁴ cells per well of a coated 96-well plate (refer to "Things to prepare in advance") in EBM-2 basal medium and all supplements.
2. After 24 hours in culture only round compact cells can be seen. Stain cells for microscope analysis at day 2.