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STEMCELL Technologies Inc rosettesep nk cell isolation kit stem cell

<t>NK</t> <t>cell</t> activation. <t>NK</t> <t>cells</t> isolated from the peripheral blood of healthy donors were stimulated for 16 h with IL-2. Activation status of the NK cells was determined by monitoring increase in the CD69 levels on the surface of the NK cells using flow cytometry. Data shown is for NK cells isolated from two different healthy donors. Control NK cells (dotted line) and IL-2 stimulated cells (solid line) were labeled with FITC-conjugated anti-CD69 antibody. Shaded area depicts binding of non-specific murine IgG antibody.

Rosettesep Nk Cell Isolation Kit Stem Cell, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Article Title: Differential expression of proteins in naïve and IL-2 stimulated primary human NK cells identified by global proteomic analysis

Journal: Journal of proteomics

doi: 10.1016/j.jprot.2013.06.024

NK cell activation. NK cells isolated from the peripheral blood of healthy donors were stimulated for 16 h with IL-2. Activation status of the NK cells was determined by monitoring increase in the CD69 levels on the surface of the NK cells using flow cytometry. Data shown is for NK cells isolated from two different healthy donors. Control NK cells (dotted line) and IL-2 stimulated cells (solid line) were labeled with FITC-conjugated anti-CD69 antibody. Shaded area depicts binding of non-specific murine IgG antibody.

Figure Legend Snippet: NK cell activation. NK cells isolated from the peripheral blood of healthy donors were stimulated for 16 h with IL-2. Activation status of the NK cells was determined by monitoring increase in the CD69 levels on the surface of the NK cells using flow cytometry. Data shown is for NK cells isolated from two different healthy donors. Control NK cells (dotted line) and IL-2 stimulated cells (solid line) were labeled with FITC-conjugated anti-CD69 antibody. Shaded area depicts binding of non-specific murine IgG antibody.

Techniques Used: Activation Assay, Isolation, Flow Cytometry, Control, Labeling, Binding Assay

Venn diagram depicting the total number of proteins identified in naïve or IL-2-activated NK cells. Human primary NK cells isolated from three healthy donors were cultured in medium supplemented with or without IL-2 for 16 h. Total proteins were extracted and digested by trypsin follow by 2D LC–MS/MS analysis. 2311 proteins were identified from naïve NK cell samples, while 2413 proteins were identified in IL-2-activated NK cells. 1999 proteins were commonly identified in both conditions.

Figure Legend Snippet: Venn diagram depicting the total number of proteins identified in naïve or IL-2-activated NK cells. Human primary NK cells isolated from three healthy donors were cultured in medium supplemented with or without IL-2 for 16 h. Total proteins were extracted and digested by trypsin follow by 2D LC–MS/MS analysis. 2311 proteins were identified from naïve NK cell samples, while 2413 proteins were identified in IL-2-activated NK cells. 1999 proteins were commonly identified in both conditions.

Techniques Used: Isolation, Cell Culture, Liquid Chromatography with Mass Spectroscopy

Validation of selected factors differentially expressed in IL-2 stimulated NK cells. Real-time-PCR analysis (a) of mRNA level of PTP1B, PCNA, and CD97 in naïve NK cells and NK cells activated by IL-2 for 16 h. Flow cytometry was used to monitor differential expression of CD48 (b), CD56 (c), CD11b (d), and CD11c (e) on naïve NK cells (dotted line) and NK cells stimulated with IL-2 (solid line) for 16 h. For each factor, data obtained for NK cells isolated from two healthy donors is shown. Non-specific IgG control is (shaded histogram) is shown only for the CD48 data.

Figure Legend Snippet: Validation of selected factors differentially expressed in IL-2 stimulated NK cells. Real-time-PCR analysis (a) of mRNA level of PTP1B, PCNA, and CD97 in naïve NK cells and NK cells activated by IL-2 for 16 h. Flow cytometry was used to monitor differential expression of CD48 (b), CD56 (c), CD11b (d), and CD11c (e) on naïve NK cells (dotted line) and NK cells stimulated with IL-2 (solid line) for 16 h. For each factor, data obtained for NK cells isolated from two healthy donors is shown. Non-specific IgG control is (shaded histogram) is shown only for the CD48 data.

Techniques Used: Biomarker Discovery, Real-time Polymerase Chain Reaction, Flow Cytometry, Quantitative Proteomics, Isolation, Control

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Journal: Journal of proteomics

Article Title: Differential expression of proteins in naïve and IL-2 stimulated primary human NK cells identified by global proteomic analysis

doi: 10.1016/j.jprot.2013.06.024

Figure Lengend Snippet: NK cell activation. NK cells isolated from the peripheral blood of healthy donors were stimulated for 16 h with IL-2. Activation status of the NK cells was determined by monitoring increase in the CD69 levels on the surface of the NK cells using flow cytometry. Data shown is for NK cells isolated from two different healthy donors. Control NK cells (dotted line) and IL-2 stimulated cells (solid line) were labeled with FITC-conjugated anti-CD69 antibody. Shaded area depicts binding of non-specific murine IgG antibody.

Article Snippet: The RosetteSep NK cell isolation kit (Stem Cell Technologies) was used and NK cell purification was conducted according to the manufacturer's protocol and the purity of the isolated NK cells was determined by monitoring of CD3, CD16, CD56, and NKp46 expression via flow cytometry as described in our previous work [ 27 – 29 ].

Techniques: Activation Assay, Isolation, Flow Cytometry, Control, Labeling, Binding Assay

Journal: Journal of proteomics

Article Title: Differential expression of proteins in naïve and IL-2 stimulated primary human NK cells identified by global proteomic analysis

doi: 10.1016/j.jprot.2013.06.024

Figure Lengend Snippet: Venn diagram depicting the total number of proteins identified in naïve or IL-2-activated NK cells. Human primary NK cells isolated from three healthy donors were cultured in medium supplemented with or without IL-2 for 16 h. Total proteins were extracted and digested by trypsin follow by 2D LC–MS/MS analysis. 2311 proteins were identified from naïve NK cell samples, while 2413 proteins were identified in IL-2-activated NK cells. 1999 proteins were commonly identified in both conditions.

Techniques: Isolation, Cell Culture, Liquid Chromatography with Mass Spectroscopy

Journal: Journal of proteomics

Article Title: Differential expression of proteins in naïve and IL-2 stimulated primary human NK cells identified by global proteomic analysis

doi: 10.1016/j.jprot.2013.06.024

Figure Lengend Snippet: Validation of selected factors differentially expressed in IL-2 stimulated NK cells. Real-time-PCR analysis (a) of mRNA level of PTP1B, PCNA, and CD97 in naïve NK cells and NK cells activated by IL-2 for 16 h. Flow cytometry was used to monitor differential expression of CD48 (b), CD56 (c), CD11b (d), and CD11c (e) on naïve NK cells (dotted line) and NK cells stimulated with IL-2 (solid line) for 16 h. For each factor, data obtained for NK cells isolated from two healthy donors is shown. Non-specific IgG control is (shaded histogram) is shown only for the CD48 data.

Techniques: Biomarker Discovery, Real-time Polymerase Chain Reaction, Flow Cytometry, Quantitative Proteomics, Isolation, Control

Journal: Journal of Leukocyte Biology

Article Title: FK506 causes cellular and functional defects in human natural killer cells

doi: 10.1189/jlb.0310148

Figure Lengend Snippet: NK cell profile of patients on FK506 monotherapy following HCT. (A) NK cell numbers in PBMCs were plotted for the healthy control (n=10) and FK506‐treated group (n=11) following unrelated BMT (UBMT; right panel table) or unrelated peripheral blood stem cell transplantation (UPBSCT; right panel table). (B) PBMCs harvested from the control or FK506‐treated group were cultured with 300 u/ml human rIL‐2 in the absence of FK506 for 7 days, and the percentage of NK cells expressing CD25 was calculated. Independent sample t test was performed by SPSS.

Article Snippet: For in vitro study, human primary NK cells were isolated from PBMCs from healthy volunteers using negative selection by the RosetteSep NK cell isolation kit (Stem Cell Technology, Vancouver, BC, Canada) and Ficoll‐Paque (Amersham Pharmacia Biotech, Korea) following the manufacturersˈ instructions.

Techniques: Transplantation Assay, Cell Culture, Expressing

Decrease in IL‐2‐stimulated NK cell proliferation by FK506. (A) Human primary NK cells were cultured with 300 u/ml human rIL‐2 in the presence of 10 ng/ml FK506 or EtOH for 7 days. Cell numbers were counted and presented as a ratio calculated by the division of a cell number in the FK506‐treated group by that of EtOH‐treated. (Left panel) Time‐dependent changes of cell number by FK506 were shown (n=7). (Right panel) The number of NK cells treated with FK506 or EtOH at the end of culture was shown (n=7). One sample t test was performed by SPSS. (B) 3H‐Thymidine incorporation assay was performed after 4 and 7 days of IL‐2 culture with/without FK506 at designated concentrations. The data shown are from 1 particular experiment representing 3 independent experiments. Error bars show sd from triplicates. (C) PI/Annexin V assay by flow cytometry was performed with cultured NK cells as above. The percentages of PI‐AnnexinV+ early apoptotic and PI+AnnexinV+ late apoptotic/necrotic NK cells were shown. The results shown are representatives of a minimum 3 independent experiments.

Journal: Journal of Leukocyte Biology

Article Title: FK506 causes cellular and functional defects in human natural killer cells

doi: 10.1189/jlb.0310148

Figure Lengend Snippet: Decrease in IL‐2‐stimulated NK cell proliferation by FK506. (A) Human primary NK cells were cultured with 300 u/ml human rIL‐2 in the presence of 10 ng/ml FK506 or EtOH for 7 days. Cell numbers were counted and presented as a ratio calculated by the division of a cell number in the FK506‐treated group by that of EtOH‐treated. (Left panel) Time‐dependent changes of cell number by FK506 were shown (n=7). (Right panel) The number of NK cells treated with FK506 or EtOH at the end of culture was shown (n=7). One sample t test was performed by SPSS. (B) 3H‐Thymidine incorporation assay was performed after 4 and 7 days of IL‐2 culture with/without FK506 at designated concentrations. The data shown are from 1 particular experiment representing 3 independent experiments. Error bars show sd from triplicates. (C) PI/Annexin V assay by flow cytometry was performed with cultured NK cells as above. The percentages of PI‐AnnexinV+ early apoptotic and PI+AnnexinV+ late apoptotic/necrotic NK cells were shown. The results shown are representatives of a minimum 3 independent experiments.

Techniques: Cell Culture, Thymidine Incorporation Assay, Annexin V Assay, Flow Cytometry

Journal: Journal of Leukocyte Biology

Article Title: FK506 causes cellular and functional defects in human natural killer cells

doi: 10.1189/jlb.0310148

Figure Lengend Snippet: Down‐regulation of adhesion molecules by FK506. (A) Cell surface expression of adhesion molecules on CD3–CD56+ NK cells was analyzed by flow cytometry after 7 days in culture with/without 10 ng/ml FK506. Shaded lines show the surface expression profile of vehicle control, and open lines show that of FK506‐treated cells. Dotted lines represent isotype control (Cont). (B) Summary of flow cytometry data shown in A. The relative amount of expression of each cell surface molecule was calculated as a ratio of mean fluorescence intensities (MFI) of the cells cultured with FK506 to those of vehicle controls. Error bars represent sd (n=5–10). One sample t test was performed by SPSS.

Techniques: Expressing, Flow Cytometry, Fluorescence, Cell Culture

Selective down‐regulation of NK activating receptors by FK506. (A) Human primary NK cells as cultured in Fig. 2 were analyzed for the surface expression of a variety of NK receptors and activation markers; n = 5–12. (B) The relative amount of expression of each cell surface marker was calculated as a ratio of mean fluorescence intensities of the cells cultured with FK506 to those of vehicle control (n=5–12). Error bars represent sd. One sample t test was performed by SPSS. (C) The relative cell population (percent) of CD25+ and CD69+ cells out of total NK cells treated with FK506 was shown (n=3). The data shown are representatives of a minimum of 3 independent experiments.

Journal: Journal of Leukocyte Biology

Article Title: FK506 causes cellular and functional defects in human natural killer cells

doi: 10.1189/jlb.0310148

Figure Lengend Snippet: Selective down‐regulation of NK activating receptors by FK506. (A) Human primary NK cells as cultured in Fig. 2 were analyzed for the surface expression of a variety of NK receptors and activation markers; n = 5–12. (B) The relative amount of expression of each cell surface marker was calculated as a ratio of mean fluorescence intensities of the cells cultured with FK506 to those of vehicle control (n=5–12). Error bars represent sd. One sample t test was performed by SPSS. (C) The relative cell population (percent) of CD25+ and CD69+ cells out of total NK cells treated with FK506 was shown (n=3). The data shown are representatives of a minimum of 3 independent experiments.

Techniques: Cell Culture, Expressing, Activation Assay, Marker, Fluorescence

Reduced natural cytotoxicity by FK506. (A) Human NK cells cultured as in Fig. 2 were subject to 51Cr release assay against K562, CEM, and U937 targets. For redirected ADCC (rADCC), effector cells were preincubated with anti‐human 2B4 prior to exposure to FcR+P815 targets. The data are representatives from 5 independent experiments, and error bars represent sd out of triplicates. (B) NK cells upon stimulation by K562 target cells were analyzed for CD107a degranulation events. The right panel shows the relative percentages of CD56+ CD107a+ populations compared with those of vehicle control (n=8). Error bar represents sd (***P<0.001). One sample t test was performed by SPSS.

Journal: Journal of Leukocyte Biology

Article Title: FK506 causes cellular and functional defects in human natural killer cells

doi: 10.1189/jlb.0310148

Figure Lengend Snippet: Reduced natural cytotoxicity by FK506. (A) Human NK cells cultured as in Fig. 2 were subject to 51Cr release assay against K562, CEM, and U937 targets. For redirected ADCC (rADCC), effector cells were preincubated with anti‐human 2B4 prior to exposure to FcR+P815 targets. The data are representatives from 5 independent experiments, and error bars represent sd out of triplicates. (B) NK cells upon stimulation by K562 target cells were analyzed for CD107a degranulation events. The right panel shows the relative percentages of CD56+ CD107a+ populations compared with those of vehicle control (n=8). Error bar represents sd (***P<0.001). One sample t test was performed by SPSS.

Techniques: Cell Culture, Release Assay

Reduced cytokine production by FK506. NK cells cultured in the presence or absence of FK506 as in Fig. 2 were mixed with K562 targets, and the production of IFN‐γ (A) and TNF‐α (B) was determined by intracellular flow cytometry. Percentages shown in the upper‐right quadrants represent (A) CD56+IFN‐γ+ or (B) CD56+TNF‐α+ NK populations (n=8 and 4, respectively). Error bars represent sd. **P < 0.01; ***P < 0.001. One sample t test was performed by SPSS.

Journal: Journal of Leukocyte Biology

Article Title: FK506 causes cellular and functional defects in human natural killer cells

doi: 10.1189/jlb.0310148

Figure Lengend Snippet: Reduced cytokine production by FK506. NK cells cultured in the presence or absence of FK506 as in Fig. 2 were mixed with K562 targets, and the production of IFN‐γ (A) and TNF‐α (B) was determined by intracellular flow cytometry. Percentages shown in the upper‐right quadrants represent (A) CD56+IFN‐γ+ or (B) CD56+TNF‐α+ NK populations (n=8 and 4, respectively). Error bars represent sd. **P < 0.01; ***P < 0.001. One sample t test was performed by SPSS.

Techniques: Cell Culture, Flow Cytometry

Down‐regulation of IL‐2R expression and reduced phosphorylation of STAT3 in FK506‐treated NK cells. (A) After a 7‐day culture of NK cells with 10 ng/ml FK506 in the presence of 300 u/ml human rIL‐2, the surface expression of α, β, and γ chains of IL‐2Rs was analyzed by flow cytometry. The data are representatives from 5 independent experiments. Closed histograms show vehicle control, open histograms show FK506‐treated NK cells, and dotted histograms show isotype control. The relative mean fluorescence intensities were calculated compared with those of vehicle control (n=10). **P < 0.01; ***P < 0.001. ANOVA and Dunnett t test were performed by SPSS. (B) Western blotting was performed with NK cells treated with FK506 for 14 h to measure the amount of total and phosphorylated STAT3 (p‐STAT3) protein. The relative intensities of the phosphorylated STAT3 were calculated as compared with those of total STAT3 (n=3; ***P<0.001).

Journal: Journal of Leukocyte Biology

Article Title: FK506 causes cellular and functional defects in human natural killer cells

doi: 10.1189/jlb.0310148

Figure Lengend Snippet: Down‐regulation of IL‐2R expression and reduced phosphorylation of STAT3 in FK506‐treated NK cells. (A) After a 7‐day culture of NK cells with 10 ng/ml FK506 in the presence of 300 u/ml human rIL‐2, the surface expression of α, β, and γ chains of IL‐2Rs was analyzed by flow cytometry. The data are representatives from 5 independent experiments. Closed histograms show vehicle control, open histograms show FK506‐treated NK cells, and dotted histograms show isotype control. The relative mean fluorescence intensities were calculated compared with those of vehicle control (n=10). **P < 0.01; ***P < 0.001. ANOVA and Dunnett t test were performed by SPSS. (B) Western blotting was performed with NK cells treated with FK506 for 14 h to measure the amount of total and phosphorylated STAT3 (p‐STAT3) protein. The relative intensities of the phosphorylated STAT3 were calculated as compared with those of total STAT3 (n=3; ***P<0.001).

Techniques: Expressing, Flow Cytometry, Fluorescence, Western Blot

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