How to Prepare a Buffy Coat from Whole Blood and Isolate PBMCs
How to generate a buffy coat from a whole blood sample, which can then be used for downstream analyses or further cell separation
A buffy coat contains leukocytes in a concentrated suspension, derived from whole blood or bone marrow. Generating a buffy coat from whole blood samples concentrates leukocytes from large sample volumes and reduces downstream cell separation handling. Additionally, the use of a buffy coat can reduce donor variability due to the elimination of donor-specific soluble serum factors from the sample.
A buffy coat can be used for downstream analyses or further processed to peripheral blood mononuclear cells (PBMCs). Then, PBMCs can be used for the isolation of specific cell populations using immunomagnetic cell isolation technologies such as ·¡²¹²õ²â³§±ð±èâ„¢. In some cases, a buffy coat can be used directly with ·¡²¹²õ²â³§±ð±èâ„¢ (see Note below).
This protocol describes how to:
- Generate a buffy coat from a whole blood sample
- Process a buffy coat to isolate PBMCs using:
Option 1: Density Gradient Centrifugation
OR
Option 2: ·¡²¹²õ²â³§±ð±èâ„¢ Direct Human PBMC Isolation Kit (Catalog #19654)
Materials
- Whole blood sample collected with anticoagulants
- Tube rack (15 mL, e.g. Catalog #200-0650; 50 mL, e.g. Catalog #200-0651)
- Tube for centrifugation
- Phosphate-buffered saline containing 2% fetal bovine serum (PBS + 2% FBS, Catalog #07905)
- Recommended medium according to your cell isolation kit's Product Information Sheet. Medium should be free of Ca++ and Mg++. Recommended options include:
- ·¡²¹²õ²â³§±ð±èâ„¢ Buffer (Catalog #20144)
- RoboSepâ„¢ Buffer (Catalog #20104)
- RoboSepâ„¢ Buffer 2 (Catalog #20164)
- Phosphate-buffered saline containing 2% fetal bovine serum and 1 mM EDTA
- Serological pipettes (25 mL, e.g. Catalog #38005; 10 mL, e.g. Catalog #38004)
- Optional: ³¢²â³¾±è³ó´Ç±è°ù±ð±èâ„¢ (Catalog #18060) or similar medium with a density of 1.077 g/mL
- Optional: ·¡²¹²õ²â³§±ð±èâ„¢ Direct Human PBMC Isolation Kit (Catalog #19654)
Protocol
Sample Preparation
Collect whole blood using an appropriate anticoagulant (such as acid-citrate-dextrose [ACD], heparin, or EDTA). Whole blood specimens may be stored at room temperature (15 - 25°C) and should be processed as soon as possible for best results.
Generate a Buffy Coat
- 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), along with a small portion of the plasma and concentrated red blood cells (RBCs). The goal 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).
- The buffy coat can be used for downstream analyses or further processed to PBMCs for the isolation of specific cell populations using immunomagnetic cell isolation technologies such as ·¡²¹²õ²â³§±ð±èâ„¢.
- 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.
For protocol modifications to use an ·¡²¹²õ²â³§±ð±èâ„¢ Direct kit with buffy coat, contact techsupport@stemcell.com.
Process a Buffy Coat to PBMCs with ³¢²â³¾±è³ó´Ç±è°ù±ð±èâ„¢ or ·¡²¹²õ²â³§±ð±èâ„¢
Option 1: ³¢²â³¾±è³ó´Ç±è°ù±ð±èâ„¢ Density Gradient Centrifugation
Watch our video tutorial: How to Isolate PBMCs from Whole Blood Using Density Gradient Centrifugation (Ficollâ„¢ or ³¢²â³¾±è³ó´Ç±è°ù±ð±èâ„¢).
This protocol can also be applied to buffy coat samples.
- Ensure all reagents are at room temperature (15 - 25°C).
- Dilute buffy coat with an equal volume of Dulbecco’s Phosphate Buffered Saline with 2% Fetal Bovine Serum (PBS + 2% FBS; Catalog #07905), or other recommended medium. Mix gently.
- Add ³¢²â³¾±è³ó´Ç±è°ù±ð±èâ„¢ (or similar density gradient medium) to a conical tube (see Table 1 for recommended volumes).
Table 1. Recommended Volumes of Buffy Coat, PBS + 2% FBS, and ³¢²â³¾±è³ó´Ç±è°ù±ð±èâ„¢ by Tube Size.
Buffy Coat (mL) PBS + 2% FBS (mL) ³¢²â³¾±è³ó´Ç±è°ù±ð±èâ„¢ (mL) Tube Size (mL) 1 1 1.5 5 2 2 3 14 3 3 3 14 4 4 4 14 5 5 10 50 10 10 15 50 15 15 15 50 - Holding the tube at a 45 degree angle, carefully layer the diluted buffy coat on top of the ³¢²â³¾±è³ó´Ç±è°ù±ð±èâ„¢. Minimize mixing of the sample with ³¢²â³¾±è³ó´Ç±è°ù±ð±èâ„¢.
- Centrifuge at 800 x g for 20 minutes at room temperature (15 - 25°C) with the brake off. If the blood or buffy coat has been stored for more than 2 hours, increase the centrifugation time to 30 minutes.
- Carefully remove and discard the upper plasma layer, leaving approximately 0.5 cm above the PBMC layer to avoid disturbing the plasma:³¢²â³¾±è³ó´Ç±è°ù±ð±èâ„¢ interface.
- Using an appropriate pipette, carefully collect the entire PBMC layer at the plasma:³¢²â³¾±è³ó´Ç±è°ù±ð±èâ„¢ interface. Include any visible PBMCs adhering to the tube wall. A small volume of the upper ³¢²â³¾±è³ó´Ç±è°ù±ð±èâ„¢ layer may be collected to ensure complete recovery of the PBMC layer, while minimizing disturbance of the erythrocyte/granulocyte pellet.
- Wash the enriched PBMCs with PBS + 2% FBS. Repeat wash.
NOTE: Centrifuging at 300 x g for 8 minutes at room temperature, with the brake on, is recommended for washing PBMCs. To remove platelets from the enriched PBMCs, perform one of the washes at 120 x g for 10 minutes at room temperature with the brake off.
- Isolated PBMCs can be used for downstream analyses or for the isolation of specific cell populations using immunomagnetic cell isolation technologies such as ·¡²¹²õ²â³§±ð±èâ„¢.
OR
Option 2: ·¡²¹²õ²â³§±ð±èâ„¢ Direct Human PBMC Isolation Kit (Catalog #19654)
Refer to the buffy coat-specific protocol described in the Product Information Sheet, which can be found on the product page under “Protocols and Documentationâ€.
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