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How to Generate a Buffy Coat

A buffy coat consists of a concentrated leukocyte suspension, originating from whole blood or bone marrow. Generation of a buffy coat from whole blood samples can be used to concentrate large sample volumes and reduce downstream cell separation handling. In addition, the use of a buffy coat can reduce donor variability due to the elimination of donor specific soluble serum factors from the sample.

Buffy coat preparation protocol

  1. Add an equal volume of recommended medium to whole blood.
    The recommended medium is indicated in the kit-specific Product Information Sheet.
    Alternatively, use phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS).
  2. Centrifuge at 800 x g for 10 minutes at room temperature (15 - 25°C) with the brake off.
  3. Remove the concentrated leukocyte band (this is the buffy coat), plus a small portion of the plasma and concentrated red blood cells (RBCs). The target is to concentrate the leukocytes approximately 5-fold while maintaining the same hematocrit (e.g. collect 2 mL of buffy coat when starting with 10 mL of whole blood).

Buffy coats can be used with many of STEMCELL Technologies’ cell separation kits:

EasySep™ Whole Blood or Buffy Coat Positive Selection kits
Follow the protocol in the kit-specific Product Information Sheet.


SepMate™
SepMate™ has been optimized for use with whole peripheral blood, cord blood and bone marrow samples. It may be used with buffy coat samples, provided that the packed RBC volume is within the indicated range:

  • SepMate™ 15: Packed RBC volume of 0.25 mL - 3 mL
  • SepMate™ 50: Packed RBC volume of 2 mL - 12 mL

RosetteSep™ and EasySep™ Direct
Although RosetteSep™ and most EasySep™ Direct kits have been optimized for use with whole peripheral blood, cells can be enriched from buffy coat provided that:

  • RBCs are present at a ratio of at least 100 RBCs per nucleated cell.
  • The concentration of nucleated cells in the sample does not exceed 5 x 10^7 cells/mL.

Note: The EasySep™ Direct HLA Crossmatch T Cell Isolation Kit (IVD) (#89671) and EasySep™ Direct HLA Crossmatch B Cell Isolation Kit (IVD) (#89684) have been optimized for isolation from whole blood, buffy coat, spleen and lymph node and are not restricted to a certain RBC ratio or cell concentration.

If you have any further questions regarding this protocol or the compatibility of buffy coats with any of our cell separation kits, please feel free to contact Product and Scientific Support at techsupport@stemcell.com.