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

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), 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).
4. 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 EasySep™.

Process a Buffy Coat to PBMCs with Lymphoprep™ or EasySep™

Option 1: Lymphoprep™ Density Gradient Centrifugation

1. Ensure all reagents are at room temperature (15 - 25°C).
2. 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.
3. Add Lymphoprep™ (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 Lymphoprep™ by Tube Size.

   Buffy Coat (mL)	PBS + 2% FBS (mL)	Lymphoprep™ (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

4. Holding the tube at a 45 degree angle, carefully layer the diluted buffy coat on top of the Lymphoprep™. Minimize mixing of the sample with Lymphoprep™.
5. 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.
6. Carefully remove and discard the upper plasma layer, leaving approximately 0.5 cm above the PBMC layer to avoid disturbing the plasma:Lymphoprep™ interface.
7. Using an appropriate pipette, carefully collect the entire PBMC layer at the plasma:Lymphoprep™ interface. Include any visible PBMCs adhering to the tube wall. A small volume of the upper Lymphoprep™ layer may be collected to ensure complete recovery of the PBMC layer, while minimizing disturbance of the erythrocyte/granulocyte pellet.
8. 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.
9. Isolated PBMCs can be used for downstream analyses or for the isolation of specific cell populations using immunomagnetic cell isolation technologies such as EasySep™.

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