Application protocol

Isolation of endothelial cells from mouse neonatal brain

The application protocol was developed to isolate high yields of viable endothelial cells from mouse neonatal brain tissue. Cells can be cultured or analyzed by flow cytometry afterwards.

Download Application | PDF 110.3 KB

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

For brain tissue dissociation

  • Neural Tissue Dissociation Kit (P) (# 130-092-628)
  • Hanks' Balanced Salt Solution (HBSS) without Ca2+ and Mg2+ (Sigma-Aldrich # 55021C) 
  • HBSS  with  Ca2+ and Mg2+ (Sigma-Aldrich  # 55037C)
  • (Optional) Beta-mercaptoethanol, 50 mM
  • 50 mL tubes
  • MACS® SmartStrainer (70 μm) (# 130-098-462) for 50 mL tubes
  • MACSmix™ Tube Rotator (# 130-090-753) in combination with an incubation oven at 37 °C
  • (Optional) gentleMACS™ Dissociator (# 130-093-235), gentleMACS Octo Dissociator (# 130-095-937), or gentleMACS Octo Dissociator with Heaters (# 130-096-427)
  • (Optional) C Tubes (#  130-093-237, # 130-096-334)
  • (Optional) MACS Neuro Medium (# 130-093-570)
  • (Optional) (Optional) MACS NeuroBrew-21 (# 130-093-566)
  • (Optional) ART® 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes.
  • (Optional) Myelin Removal Beads II, human, mouse, rat (# 130-096-433, # 130-096-733)

▲ Note: Make sure the antigen epitope that is necessary for downstream applications is conserved during the dissociation procedure. 

For a detailed list of antigen compatibilities and the right choice of NTDK refer to the table on  NTDK product page at www.miltenyibiotec.com.

In case your epitope of interest is not listed please contact technical support.  You  can also perform  a staining experiment with this antibody after using different enzyme concentrations, i.e., different dilutions of Enzyme P or T (e.g., for NTDK (P) 1:5, 1:10; for NTDK (T)  1:2.5)  prior  to  isolation experiments to analyze the stability of your antibody epitope.

For cell isolation and flow cytometry analysis

  • CD45 MicroBeads, mouse (# 130-052-301)
  • CD31 MicroBeads, mouse (# 130-097-418)
  • LD Columns (# 130-042-901) and MS Columns (# 130-042-201) and suitable MACS Separator
  • Pre-Separation Filters, 70 μm (# 130-095-823) to remove cell clumps
  • PEB buffer: Dilute MACS BSA Stock Solution (# 130‑091‑376) 1:20 with autoMACS® Rinsing Solution (# 130‑091-222). Prepare fresh.
    ▲ Note: Do not use autoMACS Running Buffer as it contains azide!
  • CD31 antibodies, mouse (clone 390) conjugated to, e.g., PE (# 130-102-608). Learn more about our antibodies and dyes.
  • MACSQuant® Analyzer 10

For cell culture

  • Fibronectin
  • EBM-2 basal medium and all supplements (Lonza, EGM™-2-MV BulletKit™, CC-3202)
Matching products:

Protocol


View timeline

This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.

Preparation of buffer for cell isolation and flow cytometry

PBE 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 1:20 with autoMACS® Rinsing Solution. Keep buffer cold (2−8 °C).

Preparation of cell culture plates

Coat the necessary 96-well culture dishes with 100 μg/mL fibronectin overnight and in an incubator at 37 °C.

  1. Dissociate mouse neonatal brain using the Neural Tissue Dissociation Kit (P), including the post-dissociation wash steps. Follow the protocol of the kit data sheet.
  2. Determine number of cells in the sample. 
  3. Centrifuge the cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.

Download data sheet

Neural Tissue Dissociation Kit (P)

Good to know

The data sheet for the Neural Tissue Dissociation Kit (P) includes a set of tips & hints to improve the quality and yield of the dissociation procedure. Refer to the data sheet appendix if, for example, the yield of viable cells is too low or a cell pellet will not form.

General notes

Notes:

  • 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.

Magnetic separation: Depletion of CD45+ cells

  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 or the autoMACS Separator with the following program: Depletes.
  6. Prepare the 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 to "Magnetic separation: Enrichment of CD31+ cells".

Magnetic separation: Enrichment of CD31+ cells


  1. Work 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 or the autoMACS 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 to "Flow cytometry analysis".
  1. Incubate cells with the chosen CD31 antibodies.
  2. Analyze cells using a flow cytometer, e.g., the MACSQuant® Analyzer 10.

For a detailed immunofluorescent staining protocol, refer to the data sheet of the chosen CD31 antibodies.


  1. Plate 5×10⁴ cells per well of a coated 96-well plate (see "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.

Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.

autoMACS, CliniMACS, the CliniMACS logo, CliniMACS Prodigy, CryoMACS, CytoMix, CytoStim, DendriMACS, ExiTron, ExpAct, FeraSpin, FeraTrack, GadoSpin, gentleMACS, LIFE 18, LIFE 21, MACS, the MACS logo, MACSductin, MACSelect, MACSfectin, MACSflex, MACSiBead, MACSiMAG, MACSmix, MACSprep, MACSQuant, MACSQuantify, MACSxpress, MidiMACS, MiniMACS, miRXplore, MultiMACS, NeuroBrew, NiraWave, OctoMACS, PepTivator, pMACS, PolySon, PrepProtect, QuadroMACS, REAfinity, REAlease, Rheo, StemMACS, StraightFrom, SuperAmp, SuperMACS, TexMACS, TheraSorb, thermoMACS, TransAct, Tyto, the Tyto logo, VarioMACS, Vio, Viobility, VioBlue, VioBright, VioGreen, Viscover, and µMACS are registered trademarks or trademarks of Miltenyi Biotec GmbH and/or its affiliates in various countries worldwide. Ficoll-Paque is a trademark of GE Healthcare companies. Vectofusin-1 is a registered trademark of Genethon. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only. Copyright © 2018 Miltenyi Biotec GmbH and/or its affiliates. All rights reserved.