Influence of NK cell magnetic bead isolation methods on phenotype and function of murine NK cells

Meinhardt, Kathrin; Kroeger, Irena; Abendroth, Alexandra; Müller, Sabine; Mackensen, Andréas; Ullrich, Evelyn

doi:10.1016/j.jim.2012.01.008

Cited by 21 publications

(22 citation statements)

References 20 publications

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“…In this context, diverse outcome could be observed when cells positively isolated by antibody bound microbeads were used for extended culture work ( Govers et al ., 2012; Greish et al ., 2012; Lapenna et al ., 2013; Meinhardt et al ., 2012). …”

Section: Discussionmentioning

confidence: 99%

Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour

Bhattacharjee¹,

Das²,

Mishra³

et al. 2017

F1000Res

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Background: Magnetic sorting of cells, based on microbead conjugated antibodies (Abs), employs positive as well as negative immunomagnetic separation methods, for isolation of a specific cell population. These microbeads are suggested to be nontoxic, biodegradable carriers conjugated to various antibodies. Isolation of cells through positive selection involves the attachment of antibody conjugated microbeads to the cells of interest, followed by their isolation in the presence of a strong magnetic field to obtain higher purity. Negative selection involves attachment of microbead conjugated antibodies to all other cell populations except the cells of interest, which remain untagged. In the present study, we compared the two methods for their effect on functional and immunophenotypic behavior of isolated CD14+ monocytes. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from blood collected from healthy volunteers by density gradient centrifugation. Human blood derived monocytes were isolated through positive selection and negative selection, making use of the appropriate monocyte isolation kit. Monocytes were then stimulated with lipopolysaccharide (LPS) and their activation and proliferation capacity were examined. The degradation or dissociation of cell-bound microbeads was also investigated. Results: We observed an impaired LPS sensitivity as well as poor activation and proliferation capacity upon stimulation by LPS in positively sorted CD14+ monocytes as compared to negatively sorted CD14+ monocytes. The attached microbeads did not degrade and remained attached to the cells even after 6 days of culture. Conclusions: Our results suggest that positively sorted CD14+ cells exhibit hampered functionality and may result in inaccurate analysis and observations in downstream applications. However, these cells can be used for immediate analytical procedures.

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Section: Discussionmentioning

confidence: 99%

Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour

Bhattacharjee¹,

Das²,

Mishra³

et al. 2017

F1000Res

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“…Mice were anesthetized by intraperitoneal injection of Inactin (thiobutabarbital, 100 mg/kg; Sigma-Aldrich; Merck Millipore) and sacrificed by cervical dislocation, and spleens were removed by mechanical dissociation and placed on a petri dish containing pre-cooled phosphate-buffered saline (PBS) supplemented with 2% FBS as described previously (12). Spleens were mashed using a moisturized cell strainer (70-µm nylon mesh) using a plunger and a cell strainer positioned above a 50-ml conical tube was used to transfer the single cell suspension through the strainer into the tube.…”

Section: Methodsmentioning

confidence: 99%

“…Cell viability was confirmed by staining the cells with 7-aminoactinomycin D (420404; 1:100; BioLegend, Inc.). NK cell purity was evaluated using flow cytometry as described previously (12). Briefly, freshly isolated NK cells or cultured NK cells were collected and washed in PBS twice.…”

Section: Methodsmentioning

confidence: 99%

“…MACS sorting is a popular method applied in areas concerning immunology, cancer research, neuroscience, and stem cell research. Through this approach, cells are positively or negatively separated, depending on specific antigens present (12). For NK cell sorting, positive selection may be gaged by selecting antibodies against NKp46 or CD49b (DX5) and negative selection may be achieved for naïve NK cell purification using commercially available kits.…”

Section: Introductionmentioning

confidence: 99%

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Comparison of the purity and vitality of natural killer cells with different isolation kits

Wang

et al. 2017

Experimental and Therapeutic Medicine

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Natural killer (NK) cells are innate lymphocytes that aid in the protection of the host from infectious diseases and cancer. In vitro studies of NK cells have provided a foundation for developing clinical adoptive NK-cell transferred immunotherapy against human tumors. To elucidate the functions and mechanisms of NK cell populations, it is important to develop an optimal, highly reproducible and reliable isolation method. The present comparative study was performed with four different NK cell isolation kits of magnetic bead labeling made by Miltenyi and Stemcell companies, including positive selection kits [cluster of differentiation (CD)-49b, using the monoclonal antibody DX5) MicroBeads] and negative selection kits. In addition, the viability of NK cells isinterleukin-2 (IL-2)-dependent in vitro and thus the concentration of IL-2 is critical for maintaining longer cell viability of NK cells. NK cell purity and viability after culturing, for 24, 48 or 72 h, with or without IL-2 (0, 100, 300 or 500 U/ml) was investigated in the present study. Purity of NK cells varied depending on the purification kit used, despite the same method being applied. Furthermore, more granulocytes were present in purified NK cells using Miltenyi sorting kits, particularly when using the negative selection kit. The main disadvantage of DX5-positive selection using the Stemcell and Miltenyi kits was that a high percentage of CD3ε+ cells were mixed into the isolated NK cells. Additionally, a significant difference of NK cell purity (P=0.003) was observed while purification was performed using different surface markers. As a consequence, the use of the positive selection kit was modified and subsequently a significantly higher purity (P=0.002) and yield (P=0.004) of NK cells was obtained. Moreover, the purity of NK cells and viability with or without a range of concentrations of IL-2 was compared. Results indicated that with a higher IL-2 concentration, the NK cell purity and viability were significantly higher (P<0.05). To our knowledge, this is the first report that has compared the disadvantages of four commercial NK cell isolation kits from two well-known companies, and identified the effect of NK cell purity and viability, using different concentrations of IL-2. To conclude, the results of the present study are fundamental in aiding the further development of NK cell therapy protocols for murine in vivo models.

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“…ELISA was performed as described by the supplier (BD Biosciences). The tumor lysis capacity of B16 melanoma cells was investigated by crystal violet assay as previously described (12).…”

Section: Methodsmentioning

confidence: 99%

Nilotinib combined with interleukin-2 mediates antitumor and immunological effects in a B16 melanoma model

et al. 2014

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Abstract. The immune system contributes to tumor cell killing which can be enhanced by cancer chemotherapeutics and immune modulatory pharmaceuticals such as tyrosine kinase inhibitors (TKIs). Recently, the beneficial effect of natural killer (NK) cells was demonstrated when combining interleukin-2 (IL-2) with the TKI imatinib. The aim of the present study was to address the antitumor and immunological effects of recently approved TKIs. Therefore, we focused on the comparison of the efficacy between imatinib and nilotinib in combination with IL-2 in a murine B16F10 melanoma model. Both TKIs possessed antitumor activity in vivo. However, the combination of nilotinib and IL-2 showed a superior outcome. Importantly, both the use of immunodeficient Rag2γc -/-mice, which lack T-lymphocytes, B-lymphocytes and NK cells, as well as NK cell-depletion in C57Bl/6 mice reduced the therapeutic effect of nilotinib. Flow cytometry revealed a significant increase in the IFN-γ-producing CD27 + NK cell subpopulation following treatment with nilotinib and IL-2. Furthermore, the therapeutic antitumor effect of nilotinib/IL-2 was completely lost in IFN-γ -/-mice. In summary, we suggest that nilotinib combined with IL-2 confers high antitumor activity involving the subset of IFN-γ-producing CD27 + NK cells. These new insights are of high importance for the understanding and development of immunotherapeutic protocols using TKIs.

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Influence of NK cell magnetic bead isolation methods on phenotype and function of murine NK cells

Cited by 21 publications

References 20 publications

Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour

Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour

Comparison of the purity and vitality of natural killer cells with different isolation kits

Nilotinib combined with interleukin-2 mediates antitumor and immunological effects in a B16 melanoma model

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