Efficient isolation of particle-free human ILC2s from peripheral blood mononuclear cells

• Published in: The Journal of immunology (1950) Vol. 200; no. 1_Supplement; pp. 120 - 120.5
• Main Authors: Poon, Grace F.T., Kyei, Stephen K., Antignano, Frann, McQueen, Karina L., Peters, Carrie E., Takei, Fumio, Valdez, Yanet, Kokaji, Andy I., Woodside, Steven M., Eaves, Allen C., Thomas, Terry E.
• Format: Journal Article
• Language: English
• Published: 01.05.2018
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.200.Supp.120.5

Abstract Group 2 innate lymphoid cells (ILC2s) have important roles in type-2 immune responses and are implicated in allergies, asthma, helminth infections and other metabolic diseases. To better understand their role in immunity, highly purified ILC2s are required. ILC2s are phenotypically defined as CD45+ lineage-negative CRTH2+ CD127+ CD161+, and comprise approximately 0.05% of peripheral blood mononuclear cells (PBMCs). Fluorescence activated cell sorting is the most common method to isolate ILC2s but it is both time-consuming and expensive because the cells are so rare. To address this issue, we have developed an immunomagnetic isolation method to obtain highly pure, particle-free ILC2s from human PBMCs in 2.5 hours. First, CRTH2 positive cells are labelled with a CRTH2-PE monoclonal antibody. These cells are then positively selected using an anti-PE antibody complex, EasySep™ Releasable RapidSpheres™ and an EasySep™ magnet. After separation, the magnetic particles are released from the positively selected cells and a second cocktail of antibody complexes and EasySep™ Dextran RapidSpheres™ is added to label non-ILC2s within the CRTH2 positive population. After a second separation, the released particles and unwanted cells are retained in the tube within the magnet, whereas the ILC2s are simply poured off and ready for use. With this method, the purity of ILC2s isolated from PBMCs was 84–95% (median 91%, n=18) with a recovery of 0.4–14.3×103 ILC2s per 108 starting PBMCs (median 2.46×103, n=18). The isolated ILC2s are functional, producing IL-13 upon in vitro stimulation with IL-33 and IL-2. Overall, EasySep™ Human ILC2 Isolation Kit allows researchers to easily and efficiently isolate highly pure and functional ILC2s.