Øren Anita, Husebø Christina, Iversen Ann-Charlotte, Austgulen Rigmor
Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Medisinsk teknisk senter, N-7489 Trondheim, Norway.
J Immunol Methods. 2005 Aug;303(1-2):1-10. doi: 10.1016/j.jim.2005.04.022.
Immunomagnetic sorting of natural killer (NK) cells from the peripheral blood of healthy donors has been evaluated in a comparative study of composition, yield and activation of target cells obtained by positive (Dynabeads, Microbeads) and negative (Microbeads) sorting procedures. Positively sorted target cells were selected by expression of the NK cell marker CD56, whereas NK cells obtained by negative sorting were those remaining after steps to remove all non-NK cell leukocyte populations were accomplished. In positive sorting, both CD56+CD3- NK cells and CD56+CD3+ natural killer T (NKT) cells were included. The NKT cell fraction differed between individuals, but not between the positive sorting methods. Whereas 20-30% of positively sorted target cells were NKT cells, only approximately 3% of negatively sorted cells were CD3+. Contamination with monocytes and B cells was low (1-3%) in all methods studied. Sorting with Microbeads (both positive and negative) gave higher cell yields than those obtained with Dynabeads (14% vs. 5% of total leukocyte numbers). A higher CD56 fluorescence intensity of NK cells and a better discrimination between the CD56bright and CD56dim NK cell subpopulations was obtained after negative sorting. Dynabeads-separated cells had, shortly after separation, a significantly higher expression (approximately 30%) of the early activation marker CD69 than cells either positively or negatively separated by Microbeads (approximately 8%). CD56+ cells positively sorted by Microbeads demonstrated a significantly higher production of TNF-alpha and IFN-gamma after IL-2 stimulation than Dynabeads-sorted cells. However, the cytotoxicity of cells obtained by the two positive sorting procedures did not differ. In conclusion, positive selection of CD56+ cells by Microbeads is better than Dynabeads, as determined from cell yield and procedure-associated cell activation and should be chosen for in vitro studies of NK/NKT cells. However, when pure NK cells and phenotypic subtypes are to be studied, negative sorting seems most appropriate.
在一项比较研究中，对通过阳性分选（Dynabeads、微珠）和阴性分选（微珠）程序获得的靶细胞的组成、产量和活化情况进行了评估，该研究评估了从健康供体外周血中免疫磁珠分选自然杀伤（NK）细胞的情况。通过NK细胞标志物CD56的表达选择阳性分选的靶细胞，而通过阴性分选获得的NK细胞是在完成去除所有非NK细胞白细胞群体的步骤后剩余的细胞。在阳性分选中，包括CD56+CD3-NK细胞和CD56+CD3+自然杀伤T（NKT）细胞。NKT细胞分数在个体之间存在差异，但在阳性分选方法之间没有差异。虽然20%-30%的阳性分选靶细胞是NKT细胞，但阴性分选细胞中只有约3%是CD3+。在所有研究方法中，单核细胞和B细胞的污染率都很低（1%-3%）。使用微珠（阳性和阴性）分选得到的细胞产量高于使用Dynabeads分选得到的细胞产量（分别为总白细胞数的14%和5%）。阴性分选后，NK细胞的CD56荧光强度更高，并且在CD56bright和CD56dim NK细胞亚群之间的区分更好。Dynabeads分选的细胞在分选后不久，早期活化标志物CD69的表达明显更高（约30%），高于通过微珠进行阳性或阴性分选的细胞（约8%）。经微珠阳性分选的CD56+细胞在IL-2刺激后TNF-α和IFN-γ的产生明显高于Dynabeads分选的细胞。然而，通过两种阳性分选程序获得细胞的细胞毒性没有差异。总之，从细胞产量以及与程序相关的细胞活化情况来看，通过微珠对CD56+细胞进行阳性选择优于Dynabeads，在NK/NKT细胞的体外研究中应选择微珠。然而，当要研究纯NK细胞和表型亚型时，阴性分选似乎最合适。
