A buffy coat contains leukocytes in a concentrated suspension, originating from whole blood or bone marrow. Generating a buffy coat from whole blood samples helps 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. This protocol describes how to generate a buffy coat from a whole blood sample, which can then be used for downstream analyses or the isolation of specific cell populations using immunomagnetic cell isolation technologies such as EasySep™.

Materials

• Whole blood sample collected with anticoagulants
• Recommended medium according to your cell isolation kit's Product Information Sheet. Recommended options include:
  • EasySep™ Buffer (Catalog #20144)
  • RoboSep™ Buffer (Catalog #20104)
  • Phosphate-buffered saline containing 2% fetal bovine serum (PBS + 2% FBS, Catalog #07905)
• Tube for centrifugation

Protocol

1. Add an equal volume of recommended medium to whole blood and mix gently.
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 an equivalent ratio of leukocytes to RBCs (e.g. collect 2 mL of buffy coat when starting with 10 mL of whole blood).

STEMCELL Technologies offers various tools and products that have been optimized for isolation of cells from whole blood, buffy coat, spleen, and lymph node, and are not restricted to a certain RBC ratio or cell concentration.

Learn more about efficient technologies for isolating cells from blood samples >