- SepMate™ tubes - 15 or 50 ml (Stemcell);
- 15 or 50 ml tubes;
- set of automatic pipettes ranging from 1 ml and 50 ml;
- centrifuge.
- Ficoll density gradient medium;
- Hanks medium;
- PBS + 0.5% BSA;
- RosetteSep™ B- or T- or Total lymphocyte enrichment coctail (Stemcell). Optional.
Figure 1. Workflow for MNCs extraction. Picture from www.stemcell.com.
The workflow for the MNCs extraction process is as follows:
- Add density gradient medium.
- Add the diluted sample by pipetting it down the side of the tube.
- Centrifuge at 1200 g for 20 minutes at room temperature.
- Pour off the interphase, which contains the enriched mononuclear cells, into a new tube.
- Wash cells with Hanks medium.
1| Add ficoll 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 ficoll will be above the insert.
Table 1. Density gradient volumes.
| SepMate™ tube | Initial blood, ml | Ficoll, ml |
|---|---|---|
| 15 | 0.5 - 4.0 | 4.5 |
| 15 | 4 - 5 | 3.5 |
| 50 | 5 - 17 | 15 |
NOTE: Small bubbles may be present in the density gradient medium after pipetting. This will not affect performance.
2| Optional step. Add appropriate volume of RosetteSep™ enrichment coctail to the blood sample. Mix gently. Incubate for 20 minutes at room temperature.
NOTE: The RosetteSep™ Enrichment Cocktail is designed to enrich lymphocytes from whole blood by negative selection. Unwanted cells are targeted for removal with Tetrameric Antibody Complexes recognizing CD16, CD36, CD66b and glycophorin A on red blood cells (RBCs). When centrifuged over a density gradient, the unwanted cells pellet along with the RBCs. The purified lymphocytes are present as a highly enriched population at the interface between the plasma and the density medium.
NOTE: This step is strongly recommended for high quality FACS sorting of small and rare lymphocyte populations.
3| Dilute sample with an equal volume of Hanks medium. Mix gently.
4| Keeping the SepMate™ tube vertical, carefully add the diluted sample by pipetting it down the side of the tube.
CRITICAL STEP: Take care not to pour the diluted sample directly through the central hole.
5| Centrifuge at 1200 g for 20 minutes at room temperature, with the brake off.
6| Aspirate the top layer (diluted plasma).
7| Pour off the interphase, which contains the enriched mononuclear cells, into a new tube.
NOTE: Some red blood cells may be present on the surface of the SepMate™ insert after centrifugation. This will not affect performance.
8| Wash enriched lymphocytes with 50 ml Hanks medium.
NOTE: Centrifuging at 400 g for 30 minutes at room temperature, with the brake on, is recommended.
9| Optional step. Repeat step 8.
10| Transfer MNCs to 1-2 ml PBS + 0.5% BSA.
NOTE: Washing with 5 ml PBS + 0.5% BSA, centrifuging at 400 g for 8 minutes at room temperature, with the brake on, is recommended.
11| Count cells.