How to Prepare a Buffy Coat from Whole Blood

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


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).
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.

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

  • Document #PR00003
  • Version 1.0.0
  • Mar 2020


  • Related Resources

    Choosing a Cell Separation Method that Meets Your Research Needs

    Learn more about the cell separation methods outlined above to choose the best method for your application.

    Compare Methods >

    Wallchart
    Frequencies of Cell Types in Human Peripheral Blood
    Video
    How EasySep™ Magnetic Cell Separation Technology Works: Fast and Easy Cell Isolation
    Video
    Cell Isolation Directly from Whole Blood without RBC Lysis or Centrifugation: EasySep™ Direct
    Video
    Simultaneous Cell Isolation from Multiple Samples Using the EasyEights™ EasySep™ Magnet

    Contact STEMCELL Technologies

    Our Customer Service, Sales, and Product and Scientific Support departments in North America are available between 6 am and 5 pm Pacific Time (9 am and 8 pm Eastern Time). One of our representatives will be happy to help you by telephone or email. Please complete the form to contact us by email. A representative will get back to you shortly.
    •  

    StemCell Technologies Inc. and affiliates ("STEMCELL Technologies") does not share your email address with third parties. StemCell Technologies Inc. will use your email address to confirm your identity and send you newsletters, transaction-related emails, promotional and customer service emails in accordance with our privacy policy. You can change your email preferences at any time.