Related Product Information
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).
- 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 www.invitrogen.com/magnets-selection 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® should be washed before use.
- Resuspend the Dynabeads® in the vial.
- Transfer the desired volume of Dynabeads® to a tube.
- Add the same volume of Buffer 1, or at least 1 ml, and mix.
- Place the tube in a magnet for 1 min and discard the supernatant.
- 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 www.invitrogen.com/cellisolation and follow our QuickLinks for recommended sample preparation procedures.
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.
|Sample volume |
(up to 108 cells/ml)
|1 ml ||1 ml |
|Volume of Dynabeads®||25 μl||50 μl|
|Total no. of cells |
processed per product
|2 x 1010 cells||1 x 1010 cells|
Positive Isolation or Depletion of Endothelial Cells from a Single Cell Suspension
- Add the appropriate volume of Dynabeads® to the prepared single cell suspension, for volumes see table 1.
- Incubate for 20 min (positive isolation) or 30 min (depletion) at 2-8°C with gentle tilting and rotation.
- Place the tube in a magnet for 2 min.
- For depletion, transfer supernatant to a new tube for further use.
- 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
- Remove medium from the culture dish with the grown endothelial cells.
- 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).
- Add 2 ml Buffer 3 (to neutralise trypsin).
- Wash once by centrifugation and resuspend endothelial cells to < 2 x 106/ml in Buffer 1.
- Add 25 μl washed Dynabeads® per ml cell suspension.
- Incubate for 20 min at 2-8°C with gentle tilting and rotation.
- Increase the volume two fold with Buffer 1 and place the tube in a magnet for 2 min.
- 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.
- 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
This product is stable until the expiry date stated on the label when stored unopened at 2-8°C. Store opened vials at 2-8°C and avoid bacterial contamination. Keep Dynabeads® in liquid suspension during storage and all handling steps, as drying will result in reduced performance. Resuspend well before use.
Warnings And Limitations
This product is for research use only. Not intended for any animal or human therapeutic or diagnostic use unless otherwise stated. Follow appropriate laboratory guidelines. This product contains 0.02% sodium azide as a preservative, which is cytotoxic.
Avoid pipetting by mouth!
Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. When disposing through plumbing drains, flush with large volumes of water to prevent azide build up. Certificate of Analysis (CoA) is available upon request. Material Safety Data Sheet (MSDS) is available at http://www.invitrogen.com.
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