Isolate or Deplete CD31+ Human Microvessel Endothelial Cells

Isolate or deplete CD31⁺ human microvessel endothelial cells directly from prepared tissue digests with Dynabeads® CD31. This product can also be used for reselection of endothelial cells grown in culture. Isolated cells are suitable for cell culture or molecular analysis. Dynabeads® CD31 will bind human umbilical vein endothelial cells (HUVEC). Microvessel endothelial cells have been isolated from many human tissues including adipose, brain, dermal, endometrial, gastric, heart, intestinal, liver, lung, placenta, renal, synovial and tonsil tissue. CD31 is expressed on all endothelial cells and also on platelets and white blood cells such as monocytes, NK cells, granulocytes, B cells and T cell subsets. Thus, Dynabeads® CD31 cannot be used directly on whole blood or bone marrow samples.

Principle of Isolation

Dynabeads® are mixed with the cell sample in a tube. The Dynabeads® will bind to the target cells during a short incubation, and then the beadbound cells are separated by a magnet.

Positive isolation - discard the supernatant and use the bead-bound cells for downstream applications. Positivel   isolated endothelial cells are pure, viable and unstimulated and are ideal for culture with the Dynabeads® still attached. Fibroblast, pericyte or smooth muscle cell contamination is avoided. The cells will grow well and adher   normally and after approximately three passages, all the beads are diluted out.

Depletion - discard the bead-bound cells and use the remaining, untouched cells for any application.

Description of Materials

Dynabeads® CD31 are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) coated with a mouse IgG1 monoclonal antibody specific for the CD31 cell surface antigen PECAM-1 (platelet endothelial cell adhesion molecule-1). The primary CD31 antibody is attached to the Dynabeads® via a secondary antibody to ensure optimal orientation of the primary antibody. The secondary antibody on the Dynabeads® is a human IgG4 anti-mouse IgG. The source of the human monoclonal antibody is free of Human Immunodeficiency Virus (HIV), Hepatitis-B Virus (HBV) and Hepatitis-C Virus (HCV).

Materials Supplied

• 5 ml Dynabeads® CD31  -  4 x 10⁸ beads/ml in phosphate buffered saline (PBS), pH 7.4, containing 0.1% bovine serum albumin (BSA) and 0.02% sodium azide (NaN₃ ). The product is sufficient for 200 tests, where one test is defined as 1 ml of cells resuspended to 1 x 10⁸ cells/ml.
• This product will process up to 2 x 10¹⁰ cells.
  

Additional Materials Required

• Magnet (Dynal® MPC™): See Dynabeads® mRNA Purification Kit for mRNA Purification from Total RNA preps for magnet recommendations.
• Mixer allowing both tilting and rotation.
• Buffer 1: PBS w/0.1% BSA, pH 7.4.
• Buffer 2: PBS (or equivalent) w/0.25% trypsin and 1 mM EDTA.
• Buffer 3: PBS w/5 % foetal calf serum (FCS).
  

Dynabeads® Washing Procedure

Dynabeads® should be washed before use.

1. Resuspend the Dynabeads® in the vial.


2. Transfer the desired volume of Dynabeads® to a tube.


3. Add the same volume of Buffer 1, or at least 1 ml, and mix.


4. Place the tube in a magnet for 1 min and discard the supernatant.


5. Remove the tube from the magnet and resuspend the washed Dynabeads® in the same volume of Buffer 1  as the initial volume of Dynabeads® (step 2).

Preparation of Single Cell Suspensions From Different Tissues (See References)

• Adipose tissue: 1, 2, 3 and 4
• Blood: 5, 6 and 7
• Bone Marrow: 8, 9, 10 and 11
• Brain tissue: 12, 13 and 14 and 15
• Dermal tissue: 16, 17, 18 and 19
• Endometrial tissue 20
• Gastric tissue: 21 and 22
• Heart tissue: 23, 24 and 25
• Human umbilical cord vein endothelial cells (HUVEC): 16, 26 and 27
• Intestinal tissue: 21 and 22
• Liver tissue: 28
• Lung tissue: 29, 30 and 31
• Placenta tissue: 32 and 33
• Renal tissue: 34
• Synovial tissue: 16, 35
• Tonsil tissue: 36

 Please visit Pan Mouse IgG - Depletion or Positive Isolation of Cells from Different Species and follow our QuickLinks for recommended sample preparation procedures.

Cell Isolation

Critical Steps for Cell Isolation

• Use a mixer that provides tilting and rotation of the tubes to ensure Dynabeads® do not settle at the bottom of the tube.
• When incubating Dynabeads® and cells, the incubation temperature must be 2-8°C to reduce phagocytic activity and other metabolic processes.
• Never use less than 25 μl Dynabeads® per ml of cell sample.
• Cell concentration can be up to 1 x 10⁸ cells per ml.

Table 1:  Volume of Dynabeads® added per ml of cell sample. The volumes can be scaled up as required.

Positive Isolation or Depletion of Endothelial Cells from a Single Cell Suspension

1. Add the appropriate volume of Dynabeads® to the prepared single cell suspension, for volumes see table 1.


2. Incubate for 20 min (positive isolation) or 30 min (depletion) at 2-8°C with gentle tilting and rotation.


3. Place the tube in a magnet for 2 min.


4. For depletion, transfer supernatant to a new tube for further use.


5. For positive isolation, discard the supernatant and wash the bead-bound cells 3 times by resuspending in Buffer 1 to the original sample volume, and separate using a magnet. The bead-bound endothelial cells are now ready for plating or further analysis.

For rapid and consistent results in protein or gene expression analysis, lyse the CD31⁺ cells while they are still attached to the beads and directly process for further molecular analysis.

Reselect Microvascular Endothelial Cells From Cultures

1. Remove medium from the culture dish with the grown endothelial cells.


2. Add 0.5 ml Buffer 2 (35 mm petri dish), incubate for 5 minutes* at 37°C, knock the dish to dislodge the cells (check by microscope).


3. Add 2 ml Buffer 3 (to neutralise trypsin).


4. Wash once by centrifugation and resuspend endothelial cells to < 2 x 10⁶/ml in Buffer 1.


5. Add 25 μl washed Dynabeads® per ml cell suspension.


6. Incubate for 20 min at 2-8°C with gentle tilting and rotation.


7. Increase the volume two fold with Buffer 1 and place the tube in a magnet for 2 min.


8. Discard the supernatant and wash the beadbound cells 3 times by resuspending in Buffer 1 to the same volume as discarded, and separate using a magnet.


9. The bead-bound endothelial cells are now ready for plating or further analysis.

* The incubation time with trypsin/EDTA may be changed to fit the user’s need.

Deplete Non-Endothelial Cells Using Dynabeads®

See references 11 and 36