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™.
Add an equal volume of recommended medium to whole blood and mix gently.
Centrifuge at 800 x g for 10 minutes at room temperature (15 - 25°C) with the brake off.
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).
Note: 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 107 cells/mL.
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.
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