RosetteSep™ is a fast and easy immunodensity procedure for the isolation of untouched cells directly from whole blood. By crosslinking unwanted cells to red blood cells (RBCs) present in the sample, target cells are purified during standard density gradient centrifugation (e.g. peripheral blood mononuclear cell (PBMC) isolation using Lymphoprep™ , Ficoll-Paque™, RosetteSep™ DM-L Density Medium or RosetteSep™ DM-M Density Medium. This approach significantly reduces sample handling time and maximizes convenience by incorporating cell subset isolation into the centrifugation process.

How Does RosetteSep™ Work?

• Unwanted cells are crosslinked to RBCs by specific antibodies forming dense immunorosettes.
• Immunorosettes pellet during density gradient centrifugation, leaving untouched and purified target cells at the interface between the plasma and the density gradient medium.
• No columns or magnets are necessary. Just incubate, spin, and collect purified cells.

The Typical RosetteSep™ Cell Enrichment Protocol

1. Add RosetteSep™ Enrichment Cocktail to whole blood and incubate for 20 minutes (10 minutes if using SepMate™ ). Dilute sample with an equal volume of PBS + 2% FBS and mix gently.
2. Layer the diluted sample on top of the density medium. Use SepMate™ to prevent the layers from mixing.
3. Centrifuge for 20 minutes at 1200 x g at room temperature, with the brake off. Use SepMate™ to reduce centrifugation time to 10 minutes with the brake on.
4. Remove the enriched cells from the density medium : plasma interface. Wash enriched cells with PBS + 2% FBS. Repeat.

Key Applications

Cell Isolation Directly From Whole Blood

The NK Cell Cytotoxicity Case Study discusses a publication from Dr. Ajay Jain’s lab at the University of Maryland School of Medicine, which used the RosetteSep™ and SepMate™ system to reduce NK cell isolation time from four hours to a single hour for a 450 mL unit of blood.