Fast and easy immunomagnetic isolation of untouched human naïve T cells in less than 15 minutes

Abstract

Abstract Peripheral blood circulating T cells can be subdivided into either antigen experienced effector and memory T cells or naïve T cells that have not yet encountered their cognate antigen. Naïve T cells are valuable tools when studying mechanisms of T cell activation and are required for foundational research into infectious diseases, cancer and transplantation. We describe here a simple immunomagnetic cell isolation protocol for the isolation of untouched human naïve T cells from fresh or previously frozen PBMC or leukapheresis samples. The column-free, negative selection procedure involves labelling and removing unwanted cells using bispecific antibody complexes that crosslink cell surface antigens to magnetic particles. The labelling cocktail is composed of antibodies that target non-T cells, memory T cells, and an optional cocktail for depleting gamma delta T cells. Briefly, the procedure involves a five minute incubation with the antibody cocktail followed by the addition of magnetic particles and two three minute magnetic separations in a hand-held EasySep™ magnet. Following the second magnetic separation, the untouched naïve T cells are simply poured off into a new tube and are ready for use. We define naïve T cells phenotypically as CD3+CD45RA+CD45RO-CD197+. Starting from fresh PBMC, we were able to obtain purities of 96.1 ± 2.3% (mean ± SD, n=14) and recoveries of 62.6% ± 24.5% (mean ± SD, n=14). Additionally, isolated naïve T cells are functional and respond by upregulating the expression of CD25 and CD69 upon stimulation. Our EasySep™ Human Naïve Pan T Cell Isolation Kit offers a fast and easy isolation method for naïve T cells that is suitable for downstream applications such as flow cytometry, cell culture or DNA/RNA extraction.