• For subsequent cell separation and cultivation it is recommended to dissociate at least 800 mg of adult mouse brain tissue.
• Volumes given below are for one adult mouse brain (max. 500 mg) in 1980 μL enzyme mix. When working with less than 500 mg, use the same volumes as indicated. When working with higher tissue quantities, scale up all reagent volumes and total volumes accordingly. A maximum of 500 mg brain tissue per gentleMACS C Tube can be processed.
• A swinging bucket rotor is recommended for centrifugation, e. g., Heraeus® Multifuge 4KR by Thermo Fisher® Scientific.

Preparation of enzyme mix

1. Enzyme P is ready to use. Prepare aliquots of appropriate volume to avoid repeated freeze-thaw-cycles. Store aliquots at –20 °C. This solution is stable for 6 months. Resuspend the lyophilized powder in the vial labeled Enzyme A with 1 mL Buffer A. Do not vortex. This solution should then be aliquoted and stored at –20 °C for later use. Avoid repeated freeze-thaw-cycles. 
2. Prepare enzyme mix 1 and enzyme mix 2 according to the table below.
    

Preparation of 1x Red Blood Cell Removal Solution

1. Dilute the Red Blood Cell Removal Solution (10×) 1:10 with double-distilled water (ddH₂O). For example, dilute 0.1 mL of cold Red Blood Cell Removal Solution (10×) with 0.9 mL cold ddH₂O.
   ▲ Note: Do not use deionized water for dilution!
2. Store the prepared 1× Red Blood Cell Removal Solution at 2–8 °C. Discard unused solution at the end of the day.
    

Coat the cell culture dishes with Human Fibronectin (Fragment). The coating concentration should be 3 μg of Human Fibronectin per cm². 

For example, when coating a 96-well plate (0.32 cm² per well):

1. Use 48 μL of a 20 μg/mL solution per well.
2. Dispense the appropriate volume of Human Fibronectin (Fragment) solution into each plate.
3. Incubate overnight at 37 °C.
4. Wash three times with ddH₂O.
5. Let the culture dish dry under sterile conditions.

• For details on the use of the gentleMACS Octo Dissociator with Heaters, refer to the user manual.
• A maximum of one mouse brain (max. 500 mg) in 2 mL enzyme mix can be processed in one gentleMACS C Tube.
• For cell culture experiments subsequent to tissue dissociation, all steps should be performed under sterile conditions.

1. Remove the mouse brain. Wash the brain in cold D-PBS.
2. Prepare the appropriate volume of enzyme mix 1 (see above) and transfer it into a gentleMACS C Tube.
3. Place the brain on a petri dish and cut it into 8 sagittal slices using a scalpel.
4. Transfer the tissue pieces into the C Tube containing 1950 μL of enzyme mix 1.
5. Transfer 30 μL of enzyme mix 2 into the C Tube.
6. Tightly close C Tube and attach it upside down onto the sleeve of the gentleMACS Octo Dissociator with Heaters.
7. Run the gentleMACS Program 37C_ABDK_01.
8. After termination of the program, detach C Tube from the gentleMACS Octo Dissociator with Heaters.
9. (Optional) Centrifuge briefly to collect the sample at the bottom of the tube.
10. Resuspend sample in D-PBS and apply it to a MACS® SmartStrainer (70 μm) placed on a 50 mL tube.
    ▲ Note: Moisten MACS SmartStrainer with D-PBS before use.
    ▲ Note: When upscaling the reagent volume and total volumes, increase also the number of MACS SmartStrainers (70 μm). One MACS SmartStrainer (70 μm) can be used for one adult mouse brain.
    ▲ Note: Cells with a diameter >70 μm may be lost. To obtain these cells within the flow-through, use a cell strainer with an appropriate mesh size.
11. Apply 10 mL of cold D-PBS onto the MACS SmartStrainer (70 μm).
12. Discard MACS SmartStrainer (70 μm) and centrifuge cell suspension at 300×g for 10 minutes at 4 °C. Aspirate supernatant completely.
13. Proceed to "Debris and red blood cell removal".
     

• Volumes given below are for the cell suspension from up to two adult mouse brains as starting material. When working with higher tissue quantities, scale up all reagent volumes accordingly.
• The cell suspension from two adult mouse brains (max. 1 g) is the maximum that can be processed in one 15 mL reagent tube.
• Always use pre-cooled buffers and solutions (4 °C).

1. Resuspend cell pellet carefully with the appropriate volume of cold D-PBS according to the table above and transfer cell suspension to a 15 mL tube. Do not vortex.
2. Add appropriate volume of cold Debris Removal Solution.
3. Mix well.
4. Overlay very gently with 4 mL of cold D-PBS.
   ▲ Note: Pipette very slowly to ensure that the D-PBS phase overlays the cell suspension and phases are not mixed.
5. Centrifuge at 4 °C and 3000×g for 10 minutes with full acceleration and full brake.
   ▲ Note: If centrifuges give suboptimal centrifugation, the acceleration and brake can be reduced.
6. Three phases are formed. Aspirate the two top phases completely and discard them.
7. Fill up with cold D-PBS to a final volume of 15 mL.
8. Gently invert the tube three times. Do not vortex!
9. Centrifuge at 4 °C and 1000×g for 10 minutes with full acceleration and full brake. Aspirate supernatant completely.
10. Resuspend cell pellet from up to two adult mouse brains carefully in 1 mL of cold 1× Red Blood Cell Lysis Solution. Do not vortex.
11. Incubate for 10 minutes in the refrigerator (2−8 °C).
12. Add 10 mL of cold D-PBS/BSA buffer.
13. Centrifuge at 4 °C and 300×g for 10 minutes. Aspirate supernatant completely.
14. Proceed to "Isolation of endothelial cells". 
     

• Work fast, keep cells cold, and use pre-cooled solutions. This will prevent capping of antibodies on the cell surface and non-specific cell labeling.
• 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 (MACS SmartStrainer (70 µm), # 130-098-462) to remove cell clumps which may clog the column. Moisten filter with buffer before use.
• 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 cell pellet in 90 µL of cold D-PBS/BSA buffer per 1×10⁷ total cells.
2. Add 10 µL of CD45 MicroBeads, mouse.
3. Mix well and incubate for 15 minutes in the dark in the refrigerator (2−8 °C).
4. Wash cells by adding 1 mL of cold D-PBS/BSA buffer per 1×10⁷ cell and centrifuge at 300×g for 5 minutes. Aspirate supernatant completely.
5. Resuspend up to 1×10⁷ cells in 500 μL of D-PBS/BSA buffer.
   ▲ Note: For higher cell numbers, scale up buffer volume accordingly.
6. (Optional) Take 20 μL for later flow cytometry analysis (original fraction 1).
7. Proceed to "Magnetic separation: Depletion of CD45⁺ cells".
    

• Choose an LD Column and an appropriate MACS Separator. For details refer to tab "Materials".
• Always wait until the column reservoir is empty before proceeding to the next step.
   

Magnetic separation with LD Columns

1. Place LD Column in the magnetic field of a suitable MACS Separator. For details refer to the respective MACS Column data sheet.
2. (Optional) Place Pre-Separation Filter (70 μm) on top of the column to remove clumps which may clog the column.
   ▲ Note: Moisten Pre-Separation Filter with buffer before use.
3. Prepare column by rinsing with 2 mL of D-PBS/BSA buffer.
4. Apply cell suspension onto the column. Collect flow-through containing unlabeled cells.
5. Wash column with 2×1 mL of D-PBS/BSA buffer. Collect unlabeled cells that pass through and combine with the flow-through from step 4. This is the target cell fraction (CD45^– cells).
   ▲ Note: Perform washing steps by adding buffer aliquots only when the column reservoir is empty.
6. (Optional, if CD45⁺ cells are needed) Remove column from the separator and place it on a suitable collection tube. Pipette 3 mL of D-PBS/BSA buffer onto the column. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column.
   ▲ Note: (Optional) Take 100 μL of positive and negative fraction for later flow cytometry analysis (pos 1 and neg 1).
7. Centrifuge at 4 °C and 300×g for 10 minutes. Aspirate supernatant completely.
8. Proceed to "Magnetic labeling of CD31⁺ cells".
    

Magnetic separation with the autoMACS® Pro Separator

• Refer to the respective user manual for instructions on how to use the autoMACS Pro Separator.
• Use D-PBS/BSA buffer. Buffers used for operating the autoMACS Pro Separator should have a temperature of ≥10 °C.

1. Prepare and prime the instrument.
2. Apply tube containing the sample and provide tubes for collecting the labeled and unlabeled cell fractions. Place sample tube in row A of the tube rack and the fraction collection tubes in rows B and C.
3. For a standard separation choose the following program: 
   Depletion: Depletes
   Collect negative fraction in row B of the tube rack. This fraction represents the target cells (CD45^– cells).
   ▲Note: (Optional) Take 100 μL of positive and negative fraction for later flow cytometric analysis (pos 1 and neg 1).
4. Centrifuge at 4 °C and 300×g for 10 minutes. Aspirate supernatant completely.
5. (Optional, if CD45⁺ cells are needed) Collect positive fraction in row C of the tube rack.
6. Proceed to "Magnetic labeling of CD31⁺ cells".
    

• Work fast, keep cells cold, and use pre-cooled solutions. This will prevent capping of antibodies on the cell surface and non-specific cell labeling.
• 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 (MACS SmartStrainer (70 µm), # 130-098-462) to remove cell clumps which may clog the column. Moisten filter with buffer before use.
• 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 cell pellet in 90 µL of cold D-PBS/BSA buffer per 1×10⁷ total cells.
2. Add 10 µL of CD31 MicroBeads, mouse.
3. Mix well and incubate for 15 minutes in the dark in the refrigerator (2−8 °C).
4. Wash cells by adding 1 mL of cold D-PBS/BSA buffer per 1×10⁷ cells and centrifuge at 300×g for 5 minutes. Aspirate supernatant completely.
5. Resuspend up to 1×10⁷ cells in 500 μL of D-PBS/BSA buffer.
   ▲ Note: For higher cell numbers, scale up buffer volume accordingly.
6. (Optional) Take 50 μL for later flow cytometric analysis (original fraction 2).
7. Proceed to "Magnetic separation: Positive selection of CD31⁺ cells".
    

Magnetic separation with MS Columns

• Choose an MS Column and MACS Separator according to the number of total cells and the number of CD31⁺ cells. For details refer to tab "Materials".
• Always wait until the column reservoir is empty before proceeding to the next step.
   

1. Place MS Column in the magnetic field of a suitable MACS Separator. For details refer to the respective MS Column data sheet.
2. (Optional) Place Pre-Separation Filter (70 μm) on top of the column to remove clumps which may clog the column.
   ▲Note: Moisten Pre-Separation Filter with buffer before use.
3. Prepare column by rinsing with 500 μL of buffer.
4. Apply cell suspension onto the column. Collect flow-through containing unlabeled cells.
5. Wash column with 3×500 μL of buffer. Collect unlabeled cells that pass through and combine with the flow‑through from step 4.
   ▲ Note: Perform washing steps by adding buffer aliquots as soon as the column reservoir is empty.
6. Remove column from the separator and place it on a suitable collection tube.
7. Pipette 1 mL of buffer onto the column. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column. This is the CD45^–CD31⁺ cell fraction.
8. To increase the purity of CD31⁺ cells, it is recommended to enrich the positive fraction over a second MS Column. Repeat the magnetic separation procedure as described in steps 1 to 7 using a new column.
   ▲Note: (Optional) Take 100 μL of positive and negative fraction for later flow cytometry analysis (pos 2 and neg 2).
9. Centrifuge at 4 °C and 300×g for 10 minutes. Aspirate supernatant completely.
10. Proceed to "Flow cytometric analysis".
     

Magnetic separation with the autoMACS Pro Separator

• Refer to the respective user manual for instructions on how to use the autoMACS Pro Separator.
• Use D-PBS/BSA buffer. Buffers used for operating the autoMACS Pro Separator should have a temperature of ≥10 °C.

1. Prepare and prime the instrument.
2. Apply tube containing the sample and provide tubes for collecting the labeled and unlabeled cell fractions. Place sample tube in row A of the tube rack and the fraction collection tubes in rows B and C.
3. For a standard separation choose the following program: 
   Positive selection: Posseld
   Collect positive fraction in row C of the tube rack. This fraction represents the target cells (CD31⁺ cells).
   ▲Note: (Optional) Take 100 μL of positive and negative fraction for later flow cytometry analysis (pos 2 and neg 2).
4. Centrifuge at 4 °C and 300×g for 10 minutes. Aspirate supernatant completely.
5. (Optional, if CD31^– cells are needed) Collect negative fraction in row B of the tube rack.
6. Proceed to "Flow cytometric analysis".
    

• Fluorochrome-conjugated Labeling Check Reagents are recommended to stain the isolated endothelial cells for flow cytometry analysis. The CD31 antibodies (clone 390) are not recommended for analysis of cells that are labeled with CD31 MicroBeads.
• The recommended antibody dilution for labeling of cells is 1:10 for up to 1×10⁶ cells/50 μL of buffer.
• Volumes given below are for up to 1×10⁶ nucleated cells. When working with fewer than 1×10⁶ 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 2×10⁶ nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).

1. (Optional) For analysis, take positive and negative fractions (pos 1, neg 1, pos 2, neg 2). Also include the original fractions 1 and 2. Centrifuge at 4 °C and 300×g for 10 minutes. Aspirate supernatant completely.
2. Resuspend up to 1×10⁶ nucleated cells per 45 μL of buffer.
3. Add 5 μL of fluorochrome-conjugated Labeling Check Reagent.
4. Mix well and incubate for 10 minutes in the dark in the refrigerator (2−8 °C).
   ▲ Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times.
5. Wash cells by adding 1 mL of D-PBS/BSA  buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
6. Resuspend cell pellet in a suitable amount of D-PBS/BSA  buffer for analysis by flow cytometry, e.g., using the MACSQuant® Analyzer 10.

1. Take a 96-well plate which has been coated overnight (refer to “Things to prepare in advance”) and plate 1×10⁵ cells in EBM-2 basal medium and all supplements.
2. After 24 hours in culture round compact cells are visible. Fix and stain cells for microscopy analysis at day 2.

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