Typical peripheral blood mononuclear cell (PBMC) isolation protocols are often laborious, requiring slow layering of blood over density gradient medium, a lengthy 30-minute centrifugation with the brake off, and careful harvesting of PBMCs using a pipette. In contrast, SepMate™ allows you to isolate PBMCs from whole blood quickly and easily, in as little as 15 minutes. The specialized SepMate™ tubes contain an insert that creates a barrier between the density gradient medium and blood sample, allowing users to quickly layer blood over the density gradient medium while preventing the layers from mixing. Centrifugation is performed with the brake on, and isolated PBMCs are simply poured off into a fresh tube.

This protocol describes how to isolate mononuclear cells from blood using SepMate™ and can also be found in the SepMate™ Product Information Sheet.

Materials

• Whole blood sample
• Density gradient medium with a density of 1.077 g/mL designed for the separation of mononuclear cells (e.g. Lymphoprep™, Catalog #07801)
• Dulbecco’s Phosphate Buffered Saline with 2% Fetal Bovine Serum (PBS + 2% FBS, Catalog #07905), or other suitable culture medium
• SepMate™ tubes

Protocol

Sample Preparation

Collect whole blood using an appropriate anticoagulant (such as acid-citrate-dextrose [ACD] or heparin). Whole blood specimens may be stored at room temperature (15 - 25°C) for no more than 48 hours before use with SepMate™.

Collect bone marrow using an appropriate anticoagulant (such as ACD or heparin). Bone marrow specimens may be stored at room temperature for no more than 48 hours before use with SepMate™.

Isolate PBMCs with SepMate™

Ensure that the sample, recommended medium (PBS + 2% FBS), density gradient medium (see Materials section above), and centrifuge are all at room temperature (15 - 25°C).

1. Add density gradient medium to the SepMate™ tube by carefully pipetting it through the central hole of the SepMate™ insert. Refer to Table 1 for required volumes. The top of the density gradient medium will be above the insert.
   NOTE: Small bubbles may be present in the density gradient medium after pipetting. These bubbles will not affect performance.

   Table 1.

   SepMate™ Tube

   Initial Sample (mL)

   Density Gradient Medium (mL)

   15

   0.5 - 4

   4.5

   15

   >4 - 5

   3.5

   50

   4 - 17

   15
2. Dilute sample with an equal volume of PBS + 2% FBS. For example, dilute 5 mL of sample with 5 mL of PBS + 2% FBS. Mix gently.
3. Keeping the SepMate™ tube vertical, add the diluted sample by pipetting it down the side of the tube. The sample will mix with the density gradient medium above the insert.
   NOTE: The sample can be poured down the side of the tube. Take care not to pour the diluted sample directly through the central hole.
4. Centrifuge at 1200 x g for 10 minutes at room temperature, with the brake on. NOTE: For samples older than 24 hours, a centrifugation time of 20 minutes is recommended.
   NOTE: Different makes and models of centrifuges may provide different rates of deceleration when braking. If a layer of MNCs is not visible following centrifugation or the recovery of MNCs is low, reduce the rate of deceleration (i.e. braking) to medium or low.
5. Pour off the top layer, which contains the enriched MNCs, into a new tube. Do not hold the SepMate™ tube in the inverted position for longer than 2 seconds.
   NOTE:

   • Some red blood cells (RBCs) may be present on the surface of the SepMate™ insert after centrifugation. These RBCs will not affect performance.
   • To reduce platelet contamination in the enriched MNCs, pipette off some of the supernatant above the MNC layer before pouring.
6. Wash enriched MNCs with PBS + 2% FBS. Repeat wash. NOTE: Centrifuging at 300 x g for 8 minutes at room temperature, with the brake on, is recommended. NOTE: To remove platelets from the enriched MNCs, perform one of the washes at 120 x g for 10 minutes at room temperature, with the brake off.
   NOTE: If the density gradient medium above the SepMate™ insert appears red after centrifugation (i.e. some RBCs have not pelleted), the SepMate™ tube can be centrifuged at 1200 x g for another 10 minutes with the brake on. This step may be necessary when processing samples that are older than 24 hours.

*SepMate™ IVD is registered as an in vitro diagnostic (IVD) device intended for the isolation of mononuclear cells from human whole blood or cord blood by density gradient centrifugation in specific regions including Canada, the United States, Europe, Australia, Brazil, and Malaysia. In all other regions, SepMate™ RUO is available for research use only. Learn more about our regulated products.