Introduction

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

Ordering Information

Sku Name Size Price Qty
11155D Dynabeads® CD31 Endothelial Cell 5 mL USD 1,070.00

Protocols

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 Depletion
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

  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

General Information

Invitrogen Dynal® AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003.

Storage/Stability

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 .
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References

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  2.    Hewett PW and Murray JC (1993) Eur J Cell Biol 62:451-454

  3.    Springhorn JP, et al. (1995) In Vitro Cell Dev Biol Anim 31:473-481

  4.    Hewett PW and Murray JC (1996) In Vitro Cell Dev Biol Anim 32:462-462 (comment to Springhorn JP, et al.)

  5.    George F, et al. (1992) Thromb Haemost 23;67:147-53

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  7.    Asahara T, et al. (1997) Science 14;275:964- 967

  8.    Masek LC and Sweetenham JW (1994) Br J Haematol 88:855-865

  9.    Rafii S, et al. (1994) Blood84:10-19
 
10.   Grosset C, et al. (1995) Blood 15;86:3763-3770

11.   Schweitzer KM, et al. (1997) Lab Invest 76:25- 36

12.   Bowman PD, et al. (1983) Ann Neurol 14:396- 402

13.   Stins MF, et al. (1997) J Neuroimmunol 76:81- 90

14.   Baev NI, et al. (1998) Stroke 29:2426-2434

15.   Aroca F, et al. (1999) J Neurooncol 43:19-25

16.   Jackson CJ, et al. (1990) J Cell Sci 96:257-262

17.   Kraling BM, et al. (1994) Lab Invest 71:745- 754

18.   Kraling BM and Bischoff J (1998) In Vitro Cell Dev Biol Anim 33:308-315

19.   Richard L, et al. (1998) Exp Cell Res 10;240:1-6.

20.   Iruela-Arispe ML, et al. (1999) Microcirculation 6:127-140

21.   Haralden G, et al. (1995) Gut 37:225-234

22.   Hull MA, et al. (1996) Gastroenterology 111:1230-240

23.   Grafe M, et al. (1993) Eur Heart J 14 Suppl I:74-81

24.   Grafe M, et al. (1994) Am J Physiol 267:H2138- 2148

25.   McDouall RM, et al. (1996) Microvasc Res. 51:137-152
 
26.   Jaffe EA, et al. (1973) J Clin Invest 52:2745- 2756

27.   Mutin M, et al. (1997) Tissue Antigens 50:449- 458

28.   Daneker GW, et al. (1998) In vitro Cell Dev Biol Anim 34:370-377

29.   Hewett PW and Murray JC (1993) Microvasc Res 46:89-102

30.   Shen J, et al. (1995) Microvasc Res 50:360-372
 
31.   Lou JN, et al. (1998) In Vitro Cell Dev Biol Anim 34:529-536

32.   Drake BL and Loke YW (1991) Hum Reprod 6:1156-1159

33.   Kacemi A, et al. (1996) Cell Tissue Res 283:183-190

34.   Martin M, et al. (1997) In Vitro Cell Dev BiolAnim 33: 261-269

35.   Abbot SE, et al. (1992) Arthritis Rheum 35:401-406

36.   Baekkevold ES, et al. (1999) Lab Invest 79: 327-336

37.   Simmons DL, et al. (1990) J Exp Med. 1;171: 2147-2152

38.   Newman PJ, et al. (1990) Science .9;247:1219- 1222

39.   Kuzu I, et al. (1992) J Clin Pathol 45:143-148

40.   Garlanda C and Dejana E (1997) Arterioscler Thromb Vasc Biol 17:1193-1202

41.   Hewett PW and Murray JC (1993) In Vitro Cell Dev Biol Anim 29A:823-830

42.   Conrad-Lapostolle V, et al. (1996) Cell Biol Toxicol 12:189-197

43.   Wang X, et al. (2003) Am J Obstet Gynecol.189:5:1445-51

44.   Liu YB, et al. (2003) World J Gastroenterol. 11:2419-23

45.   Tiwari A, et al. (2003) Biotechnol Appl Biochem. 38:35-41

46. Xaymardan M, et al. (2004) J Exp Med. 15; 199 (6):797-804
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111.55D.indd     Rev 003      5-May-2007