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Dextran Sedimentation of Erythrocytes

  1. Draw 15 ml blood into a 20 ml syringe containing 0.6 ml 15% Dextran and 0.6 ml 0.25M EDTA (both in 0.9% saline at room temperature).
  2. Place syringe at 30° angle for 10-30 minutes, then stand upright for another 15-30 minutes at room temperature.
  3. With a new, bent 18 gauge needle (Monoject #8881-200078), carefully push the leukocyte-rich plasma into a 15 ml centrifuge tube.
  4. Centrifuge at 300 x g for 5-10 minutes at room temperature.
  5. Aspirate supernatant and resuspend pelleted cells with 1 ml protein-free buffer (eg. Phosphate Buffered Saline, PBS).
  6. Lyse erythrocytes for 10 seconds with 9 ml H2O, then add 1 ml of 10X PBS.

OR use BDIS protocol (hypotonic NH4Cl/KHCO3/EDTA buffer) for lysis instead:

  1. To 1 ml cells add 14 ml of 1X Lysing Solution. See Becton Dickinson Source Book Procedures Section 2.11 (or our online protocol) for recipe.
  2. Mix well and incubate 3-5 minutes at room temperature.
  1. Centrifuge at 300 x g 5 minutes at room temperature.
  2. Aspirate supernatant, resuspend cell pellet with 5 ml of COLD buffer.
  3. Centrifuge at 300 x g 5 minutes at 2° to 8°C.
  4. Aspirate supernatant, resuspend to 1 ml buffer with protein carrier (eg. Bovine Serum Albumin, BSA).

References

  1. Tausk F, Fey, M, Gigli, I. J Immumol 143:3295-3302. 1989.
  2. Moore, JS, Caulkins CE. J Immounol 134:3838-3844, 1985.
  3. Becton Dickinson Monoclonal Antibodies Source Book, 1995.