Dextran Sedimentation of Erythrocytes

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Dextran Sedimentation of Erythrocytes

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).

Place syringe at 30° angle for 10-30 minutes, then stand upright for another 15-30 minutes at room temperature.

With a new, bent 18 gauge needle (Monoject #8881-200078), carefully push the leukocyte-rich plasma into a 15 ml centrifuge tube.

Centrifuge at 300 x g for 5-10 minutes at room temperature.

Aspirate supernatant and resuspend pelleted cells with 1 ml protein-free buffer (eg. Phosphate Buffered Saline, PBS).

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:

To 1 ml cells add 14 ml of 1X Lysing Solution. See Becton Dickinson Source Book Procedures Section 2.11 (which has been updated and is available online from the BD site at BDIS or our online protocol for recipe.)

Mix well and incubate 3-5 minutes at room temperature.

Centrifuge at 300 x g 5 minutes at room temperature.
Aspirate supernatant, resuspend cell pellet with 5 ml of COLD buffer.
Centrifuge at 300 x g 5 minutes at 2° to 8°C.
Aspirate supernatant, resuspend to 1 ml buffer with protein carrier (eg. Bovine Serum Albumin, BSA).

References:

Tausk F, Fey, M, Gigli, I. J Immumol 143:3295-3302. 1989.

Moore, JS, Caulkins CE. J Immounol 134:3838-3844, 1985.

Becton Dickinson Monoclonal Antibodies Source Book, 1995.

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Dennis J. Young, UCSD Cancer Center Flow Cytometry Core Facility Dextran Sedimentation