We have tried to purify NK-cells using MACS-columns and the NK-cell untouch kit from Miltenyi. This works pretty well, however, if there are still some contaminating T-cells (even
Hi, Marleen.. Is there particular reason to use IL-2? One of the reasones T cells grow faster could be IL-2 as they expressed higer receptor(s), a, b, g. It is well konwn that IL-15 is important at the early stage of NK cell differentiation although T cells will also responde to IL-15 as well (perhaps less than IL-2). Otherwise you should use sorter (FACSAria).. I feel that you have a reason to use untouch KIT (even though the sorting gives a high purity).. Good luck ..
It's best to start with freshly isolated PBMCs. I would stain with CD3 and CD56, facs sort at low pressure (15psi) to avoid shearing of the NK cells and then plate into pre-prepared feeder cell cultures (we normally use a 1: 1 mix of irradiated (40 Greys) Daudi cells and RPMI 8866 cells) with human serum (male AB-, 10%) and IL-2 (50U/ml). For plating the NK cells we normally use a ratio of 1 feeder cell per 3 NK cells. Leave for 5-7 days and then stain for CD3 and CD56. You can modify the cocktail of antibodies if it is of interest to sort particular subpopulations of NK cells.