human nk cell line nk92  (ATCC)


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    ATCC human nk cell line nk92
    Engineering and establishment of <t>α-Cot-NK92</t> cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Human Nk Cell Line Nk92, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 1 article reviews
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    human nk cell line nk92 - by Bioz Stars, 2024-04
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    1) Product Images from "A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets"

    Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2022.1089369

    Engineering and establishment of α-Cot-NK92 cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: Engineering and establishment of α-Cot-NK92 cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Techniques Used: Expressing, Cell Culture, Transduction, Flow Cytometry, Incubation

    The action of α-Cot-NK92 cells depends on the type of conjugator. (A) Expression levels of HER2 and EGFR in various tumor cell lines, including mammary gland adenocarcinoma (AU565), ovarian adenocarcinoma (SK-OV-3), and epidermoid carcinoma (A431), were confirmed through FACS. Histograms are representative of at least three independent experiments. (B) Cytotoxicity against HER2 + , EGFR + , or HER2 + and EGFR + tumor cells mediated by α-Cot-NK92 cells with or without α-HER2-Cot or ZEGFR-Cot. The α-Cot-NK92 cells were co-cultured with calcein-stained tumor cells with or without a conjugator for 4 h at E:T ratios of 5:1, 1:1, and 0.5:1. The intensity of fluorescence emitted from the lysed target cells was measured using a fluorescence plate reader (SpectraMax i3x). All cytotoxicity data are represented as the mean ± S.D. of triplicate experiments. The statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05 vs. vehicle; ns: not significant. (C, D) IFN-γ and TNF-α secretion and CD107a expression in α-Cot-NK92 cells cultured with or without HER2 + EGFR + AU565 (C) or HER2 − EGFR + A431 (D) cells and with/without the conjugator (α-HER2-Cot or ZEGFR-Cot). Secreted IFN-γ and TNF-α levels were measured via ELISA using the medium of α-Cot-NK92 cells co-cultured with target cells at a 1:1 E:T ratio for 12 h. All experiments were performed in triplicate wells for each condition and repeated at least two times. Average values are shown as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t -test. *** P < 0.001; ** P < 0.01; ns: not significant. CD107a expression in α-Cot-NK92 cells was evaluated through FACS analysis after co-culture with target cells at a 5:1 E:T ratio for 4 h. Each value represents the percentage of CD56 + CD107a + cells in flow cytometric density plots. (E) Expression level of HER2 and EGFR in pulmonary adenocarcinoma (A549) confirmed through FACS analysis. Histograms are representative of at least three independent experiments. (F) Kinetics of α-Cot-NK92 cell-mediated tumor cell lysis using the xCELLigence real time cell analysis system. NK92 or α-Cot-NK92 cells were co-cultured with unlabeled A549 cells with/without ZEGFR-Cot at a 5:1 E:T ratio and monitored over time. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: The action of α-Cot-NK92 cells depends on the type of conjugator. (A) Expression levels of HER2 and EGFR in various tumor cell lines, including mammary gland adenocarcinoma (AU565), ovarian adenocarcinoma (SK-OV-3), and epidermoid carcinoma (A431), were confirmed through FACS. Histograms are representative of at least three independent experiments. (B) Cytotoxicity against HER2 + , EGFR + , or HER2 + and EGFR + tumor cells mediated by α-Cot-NK92 cells with or without α-HER2-Cot or ZEGFR-Cot. The α-Cot-NK92 cells were co-cultured with calcein-stained tumor cells with or without a conjugator for 4 h at E:T ratios of 5:1, 1:1, and 0.5:1. The intensity of fluorescence emitted from the lysed target cells was measured using a fluorescence plate reader (SpectraMax i3x). All cytotoxicity data are represented as the mean ± S.D. of triplicate experiments. The statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05 vs. vehicle; ns: not significant. (C, D) IFN-γ and TNF-α secretion and CD107a expression in α-Cot-NK92 cells cultured with or without HER2 + EGFR + AU565 (C) or HER2 − EGFR + A431 (D) cells and with/without the conjugator (α-HER2-Cot or ZEGFR-Cot). Secreted IFN-γ and TNF-α levels were measured via ELISA using the medium of α-Cot-NK92 cells co-cultured with target cells at a 1:1 E:T ratio for 12 h. All experiments were performed in triplicate wells for each condition and repeated at least two times. Average values are shown as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t -test. *** P < 0.001; ** P < 0.01; ns: not significant. CD107a expression in α-Cot-NK92 cells was evaluated through FACS analysis after co-culture with target cells at a 5:1 E:T ratio for 4 h. Each value represents the percentage of CD56 + CD107a + cells in flow cytometric density plots. (E) Expression level of HER2 and EGFR in pulmonary adenocarcinoma (A549) confirmed through FACS analysis. Histograms are representative of at least three independent experiments. (F) Kinetics of α-Cot-NK92 cell-mediated tumor cell lysis using the xCELLigence real time cell analysis system. NK92 or α-Cot-NK92 cells were co-cultured with unlabeled A549 cells with/without ZEGFR-Cot at a 5:1 E:T ratio and monitored over time. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Techniques Used: Expressing, Cell Culture, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Lysis

    Combination with conjugator enhances recognition of multiple antigens by α-Cot-NK92 cells. (A) Additive cytotoxic effects of α-Cot-NK92 cells in multi-targeting through co-treatment with α-HER2-Cot and ZEGFR-Cot. α-Cot-NK92 cells were co-cultured with calcein-stained AU565 cells with/without α-HER2-Cot (50 or 500 μg/mL), ZEGFR-Cot (5 or 50 ng/mL), or both α-HER2-Cot and ZEGFR-Cot at a 1:1 E:T ratio for 4 h, or were co-cultured with calcein-stained A549-Red-Fluc cells with/without α-HER2-Cot (5 or 500 μg/mL) and/or ZEGFR-Cot (2.5 or 50 ng/mL) at a 5:1 E:T ratio for 4 h. (B) To mimic the heterogeneity of the recurrent tumor, the EGFR + HER2 − MDA-MB-231 and EGFR − HER2 + MDA-MB-453 cells were mixed. On day 1, one group was not treated with α-Cot-NK92 cells (none) and the other groups were co-cultured with α-Cot-NK92 cells with/without ZEGFR-Cot at a 1:1 E:T ratio for 4 h. After removing α-Cot-NK92 cells, the remaining target cells were cultured for 2 d and then co-cultured with the α-Cot-NK92 cells with ZEGFR-cot or α-HER2-cot. (C) Cytotoxicity of α-Cot-NK92 cells to tumor cells on day 1 and (D) after 2 d in a model mimicking a recurrent tumor. Population change was measured using a FACS. All cytotoxicity data are presented as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer. α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92.
    Figure Legend Snippet: Combination with conjugator enhances recognition of multiple antigens by α-Cot-NK92 cells. (A) Additive cytotoxic effects of α-Cot-NK92 cells in multi-targeting through co-treatment with α-HER2-Cot and ZEGFR-Cot. α-Cot-NK92 cells were co-cultured with calcein-stained AU565 cells with/without α-HER2-Cot (50 or 500 μg/mL), ZEGFR-Cot (5 or 50 ng/mL), or both α-HER2-Cot and ZEGFR-Cot at a 1:1 E:T ratio for 4 h, or were co-cultured with calcein-stained A549-Red-Fluc cells with/without α-HER2-Cot (5 or 500 μg/mL) and/or ZEGFR-Cot (2.5 or 50 ng/mL) at a 5:1 E:T ratio for 4 h. (B) To mimic the heterogeneity of the recurrent tumor, the EGFR + HER2 − MDA-MB-231 and EGFR − HER2 + MDA-MB-453 cells were mixed. On day 1, one group was not treated with α-Cot-NK92 cells (none) and the other groups were co-cultured with α-Cot-NK92 cells with/without ZEGFR-Cot at a 1:1 E:T ratio for 4 h. After removing α-Cot-NK92 cells, the remaining target cells were cultured for 2 d and then co-cultured with the α-Cot-NK92 cells with ZEGFR-cot or α-HER2-cot. (C) Cytotoxicity of α-Cot-NK92 cells to tumor cells on day 1 and (D) after 2 d in a model mimicking a recurrent tumor. Population change was measured using a FACS. All cytotoxicity data are presented as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer. α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92.

    Techniques Used: Cell Culture, Staining

    α-Cot-NK92 cells acts in a conjugator-dependent manner at the immune synapse (A) Images depicting the sequential steps in the interaction between α-Cot-NK92 and AU565 cells with or without conjugators (α-HER2-Cot and ZEGFR-Cot). The α-Cot-NK92 and AU565 (HER2 + EGFR + ) cells were labeled with LysoSensor Green and DDAO-SE, respectively, and co-cultured in a medium containing propidium iodide to assess the lytic dynamics of α-Cot-NK92 cells with or without conjugators. Images were acquired using a fluorescence microscope. The snapshots are provided in Supplementary Mov1. (B) Percentage of α-Cot-NK92 cells remaining at each step after 2, 4, or 6 h of co-incubation with AU565 cells with/without conjugators. (C) Duration of the process, and (D) ratio of α-Cot-NK92 cells that kill target cells for target-contacting cells. Data are shown as the mean (red line) ± S.D. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001. Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: α-Cot-NK92 cells acts in a conjugator-dependent manner at the immune synapse (A) Images depicting the sequential steps in the interaction between α-Cot-NK92 and AU565 cells with or without conjugators (α-HER2-Cot and ZEGFR-Cot). The α-Cot-NK92 and AU565 (HER2 + EGFR + ) cells were labeled with LysoSensor Green and DDAO-SE, respectively, and co-cultured in a medium containing propidium iodide to assess the lytic dynamics of α-Cot-NK92 cells with or without conjugators. Images were acquired using a fluorescence microscope. The snapshots are provided in Supplementary Mov1. (B) Percentage of α-Cot-NK92 cells remaining at each step after 2, 4, or 6 h of co-incubation with AU565 cells with/without conjugators. (C) Duration of the process, and (D) ratio of α-Cot-NK92 cells that kill target cells for target-contacting cells. Data are shown as the mean (red line) ± S.D. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001. Cot, cotinine; NK, natural killer.

    Techniques Used: Labeling, Cell Culture, Fluorescence, Microscopy, Incubation

    α-Cot-NK92 cells with a conjugator inhibit tumor growth in an in vivo lung cancer model. (A) The schedule of in vivo studies using luciferase-expressing A549-Red-Fluc cells in mouse metastasis model treated with α-Cot-NK92 cells with or without ZEGFR-Cot. (B) Representative in vivo bioluminescence imaging of each group treated with α-Cot-NK92 cells with or without ZEGFR-Cot and Taxol (10 mg/kg body weight) compared to levels in the untreated group (none) on day 57 after tumor cell injection. (C) The tumor burden of each group (n = 10) in in vivo lungs quantified as total flux (photon/s), which was monitored weekly for 57 d after the A549-Red-Fluc injection. (D, E) Representative BLI (D) and total quantitative flux (E) in ex vivo lungs of each group extracted on day 57 after in vivo BLI. *** P < 0.001 vs. none; * P < 0.05 vs. vehicle; Tukey’s multiple comparison test; n.s.: not significant. Bioluminescent images were acquired after an i.p. injection of D -luciferin (150 mg/kg body weight) and analyzed using the Living Image Software. Total flux data were plotted as mean ± SD. Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: α-Cot-NK92 cells with a conjugator inhibit tumor growth in an in vivo lung cancer model. (A) The schedule of in vivo studies using luciferase-expressing A549-Red-Fluc cells in mouse metastasis model treated with α-Cot-NK92 cells with or without ZEGFR-Cot. (B) Representative in vivo bioluminescence imaging of each group treated with α-Cot-NK92 cells with or without ZEGFR-Cot and Taxol (10 mg/kg body weight) compared to levels in the untreated group (none) on day 57 after tumor cell injection. (C) The tumor burden of each group (n = 10) in in vivo lungs quantified as total flux (photon/s), which was monitored weekly for 57 d after the A549-Red-Fluc injection. (D, E) Representative BLI (D) and total quantitative flux (E) in ex vivo lungs of each group extracted on day 57 after in vivo BLI. *** P < 0.001 vs. none; * P < 0.05 vs. vehicle; Tukey’s multiple comparison test; n.s.: not significant. Bioluminescent images were acquired after an i.p. injection of D -luciferin (150 mg/kg body weight) and analyzed using the Living Image Software. Total flux data were plotted as mean ± SD. Cot, cotinine; NK, natural killer.

    Techniques Used: In Vivo, Luciferase, Expressing, Imaging, Injection, Ex Vivo, Software

    Monoclonal α-Cot-NK92 cell lines have different killing potentials. (A, B) α-Cot-NK92 clones were established through single cell sorting using FACS in an expression level-dependent manner for CAR-Myc. Black triangles with a line indicate the expression level of CAR-Myc. The ability of α-Cot-NK92 clones to lyse AU565 cells was assessed using co-culture with α-HER2-Cot (A: 500 ng/mL) or ZEGFR-Cot (B: 50 ng/mL) for 4 h at E:T ratios of 5:1, 2:1, and 1:1. Ve: vehicle. (C, D) Comparison of cytolytic potency between low (L8) and medium (M2) CAR-expressing α-Cot-NK92 clones through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells in a dose-dependent manner of α-HER2-Cot (C: from a concentration of 500 to 62.5 ng/mL, upper panel) or ZEGFR-Cot (D: from a concentration of 12.5 to 1.56 ng/mL, lower panel). (E) Comparison of cytolytic potency between M2 clone and heterogeneous α-Cot-NK92 cells through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells with α-HER2-Cot (125 ng/mL) or ZEGFR-Cot (6.25 ng/mL). (F) Relative expression level (%) and mean fluorescence intensity (MFI) of HER2 and EGFR in breast cancer cells (AU565) and normal lung cells (WI-38). (G) Cytolytic activity of NK92, monoclonal α-Cot-NK92-L8, and α-Cot-NK92-M2 cells with ZEGFR-Cot or HER2-Cot against AU565 and WI-38 cells. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: Monoclonal α-Cot-NK92 cell lines have different killing potentials. (A, B) α-Cot-NK92 clones were established through single cell sorting using FACS in an expression level-dependent manner for CAR-Myc. Black triangles with a line indicate the expression level of CAR-Myc. The ability of α-Cot-NK92 clones to lyse AU565 cells was assessed using co-culture with α-HER2-Cot (A: 500 ng/mL) or ZEGFR-Cot (B: 50 ng/mL) for 4 h at E:T ratios of 5:1, 2:1, and 1:1. Ve: vehicle. (C, D) Comparison of cytolytic potency between low (L8) and medium (M2) CAR-expressing α-Cot-NK92 clones through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells in a dose-dependent manner of α-HER2-Cot (C: from a concentration of 500 to 62.5 ng/mL, upper panel) or ZEGFR-Cot (D: from a concentration of 12.5 to 1.56 ng/mL, lower panel). (E) Comparison of cytolytic potency between M2 clone and heterogeneous α-Cot-NK92 cells through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells with α-HER2-Cot (125 ng/mL) or ZEGFR-Cot (6.25 ng/mL). (F) Relative expression level (%) and mean fluorescence intensity (MFI) of HER2 and EGFR in breast cancer cells (AU565) and normal lung cells (WI-38). (G) Cytolytic activity of NK92, monoclonal α-Cot-NK92-L8, and α-Cot-NK92-M2 cells with ZEGFR-Cot or HER2-Cot against AU565 and WI-38 cells. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Techniques Used: Clone Assay, FACS, Expressing, Co-Culture Assay, Concentration Assay, Fluorescence, Activity Assay

    human nk cell line nk92  (ATCC)


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    ATCC human nk cell line nk92
    Human Nk Cell Line Nk92, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human nk cell line nk92/product/ATCC
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human nk cell line nk92 - by Bioz Stars, 2024-04
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    human nk cell line nk92 mi  (ATCC)


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    ATCC human nk cell line nk92 mi
    Human Nk Cell Line Nk92 Mi, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human nk cell line nk92 mi/product/ATCC
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human nk cell line nk92 mi - by Bioz Stars, 2024-04
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    human nk cell line nk92 cells  (ATCC)


    Bioz Verified Symbol ATCC is a verified supplier
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    ATCC human nk cell line nk92 cells
    CBX3 was identified as a downstream effector to be involved in TYMSOS-regulated breast cancer progression (A and B) The mRNA level of CBX3 in breast tumors (n = 58) and their normal counterparts (n = 58) were detected by RT-qPCR. (C) Pearson correlation analysis between TYMSOS and CBX3 in breast tumors. (D) Data analyses of CBX3 expression in breast cancer based on UALCAN database ( https://ualcan.path.uab.edu/index.html ). (E) The expression of CBX3 in MCF10A and breast cancer cells (Hs578T, T47D, MCF-7, MDA-MB-231, and MDA-MB-468 cells) were detected by RT-qPCR and Western blot, respectively. (F) The protein level of CBX3 in CBX3-knockdown MDA-MB-231 or CBX3-overexpressing MCF-7 cells was detected by Western blot. (G) Cell proliferation of transfected breast cancer cells was assessed by CCK-8 assay. (H) The colony-forming ability of transfected breast cancer cells was monitored by colony formation assay. (I) Cell migration and invasion of transfected breast cancer cells were detected by Transwell assays. Scale bar, 100 μm. Activated <t>NK92</t> cells were co-cultured with transfected breast cancer cells. (J) Cytotoxicity of NK92 cells was detected by CytoTox96 cytotoxicity assay. (K) The secreted IFN-γ and TNF-α in cell culture supernatant of co-culture system were detected by ELISA assay. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.
    Human Nk Cell Line Nk92 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human nk cell line nk92 cells/product/ATCC
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human nk cell line nk92 cells - by Bioz Stars, 2024-04
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    1) Product Images from "LncRNA TYMSOS facilitates breast cancer metastasis and immune escape through downregulating ULBP3"

    Article Title: LncRNA TYMSOS facilitates breast cancer metastasis and immune escape through downregulating ULBP3

    Journal: iScience

    doi: 10.1016/j.isci.2023.107556

    CBX3 was identified as a downstream effector to be involved in TYMSOS-regulated breast cancer progression (A and B) The mRNA level of CBX3 in breast tumors (n = 58) and their normal counterparts (n = 58) were detected by RT-qPCR. (C) Pearson correlation analysis between TYMSOS and CBX3 in breast tumors. (D) Data analyses of CBX3 expression in breast cancer based on UALCAN database ( https://ualcan.path.uab.edu/index.html ). (E) The expression of CBX3 in MCF10A and breast cancer cells (Hs578T, T47D, MCF-7, MDA-MB-231, and MDA-MB-468 cells) were detected by RT-qPCR and Western blot, respectively. (F) The protein level of CBX3 in CBX3-knockdown MDA-MB-231 or CBX3-overexpressing MCF-7 cells was detected by Western blot. (G) Cell proliferation of transfected breast cancer cells was assessed by CCK-8 assay. (H) The colony-forming ability of transfected breast cancer cells was monitored by colony formation assay. (I) Cell migration and invasion of transfected breast cancer cells were detected by Transwell assays. Scale bar, 100 μm. Activated NK92 cells were co-cultured with transfected breast cancer cells. (J) Cytotoxicity of NK92 cells was detected by CytoTox96 cytotoxicity assay. (K) The secreted IFN-γ and TNF-α in cell culture supernatant of co-culture system were detected by ELISA assay. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.
    Figure Legend Snippet: CBX3 was identified as a downstream effector to be involved in TYMSOS-regulated breast cancer progression (A and B) The mRNA level of CBX3 in breast tumors (n = 58) and their normal counterparts (n = 58) were detected by RT-qPCR. (C) Pearson correlation analysis between TYMSOS and CBX3 in breast tumors. (D) Data analyses of CBX3 expression in breast cancer based on UALCAN database ( https://ualcan.path.uab.edu/index.html ). (E) The expression of CBX3 in MCF10A and breast cancer cells (Hs578T, T47D, MCF-7, MDA-MB-231, and MDA-MB-468 cells) were detected by RT-qPCR and Western blot, respectively. (F) The protein level of CBX3 in CBX3-knockdown MDA-MB-231 or CBX3-overexpressing MCF-7 cells was detected by Western blot. (G) Cell proliferation of transfected breast cancer cells was assessed by CCK-8 assay. (H) The colony-forming ability of transfected breast cancer cells was monitored by colony formation assay. (I) Cell migration and invasion of transfected breast cancer cells were detected by Transwell assays. Scale bar, 100 μm. Activated NK92 cells were co-cultured with transfected breast cancer cells. (J) Cytotoxicity of NK92 cells was detected by CytoTox96 cytotoxicity assay. (K) The secreted IFN-γ and TNF-α in cell culture supernatant of co-culture system were detected by ELISA assay. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.

    Techniques Used: Quantitative RT-PCR, Expressing, Western Blot, Transfection, CCK-8 Assay, Colony Assay, Migration, Cell Culture, Cytotoxicity Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

    TYMSOS promoted the malignant phenotypes of breast cancer cells (A) The TYMSOS level in MCF10A and breast cancer cells (Hs578T, T47D, MCF-7, MDA-MB-231, and MDA-MB-468 cells) was detected by RT-qPCR. (B) The TYMSOS level in TYMSOS-knockdown MDA-MB-231 or TYMSOS-overexpressing MCF-7 cells were detected by RT-qPCR. (C) Cell proliferation of transfected breast cancer cells was assessed by CCK-8 assay. (D) The colony-forming ability of transfected breast cancer cells was monitored by colony formation assay. (E) Cell migration and invasion of transfected breast cancer cells were detected by Transwell assays. Scale bar, 100 μm. Activated NK92 cells were co-cultured with transfected breast cancer cells. (F) Cytotoxicity of NK92 cells was detected by CytoTox96 cytotoxicity assay. (G) The secreted IFN-γ and TNF-α in cell culture supernatant of co-culture system were detected by ELISA assay. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.
    Figure Legend Snippet: TYMSOS promoted the malignant phenotypes of breast cancer cells (A) The TYMSOS level in MCF10A and breast cancer cells (Hs578T, T47D, MCF-7, MDA-MB-231, and MDA-MB-468 cells) was detected by RT-qPCR. (B) The TYMSOS level in TYMSOS-knockdown MDA-MB-231 or TYMSOS-overexpressing MCF-7 cells were detected by RT-qPCR. (C) Cell proliferation of transfected breast cancer cells was assessed by CCK-8 assay. (D) The colony-forming ability of transfected breast cancer cells was monitored by colony formation assay. (E) Cell migration and invasion of transfected breast cancer cells were detected by Transwell assays. Scale bar, 100 μm. Activated NK92 cells were co-cultured with transfected breast cancer cells. (F) Cytotoxicity of NK92 cells was detected by CytoTox96 cytotoxicity assay. (G) The secreted IFN-γ and TNF-α in cell culture supernatant of co-culture system were detected by ELISA assay. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.

    Techniques Used: Quantitative RT-PCR, Transfection, CCK-8 Assay, Colony Assay, Migration, Cell Culture, Cytotoxicity Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

    Silencing of TYMSOS repressed tumor growth and boosted NK cell sensitivity in vivo (A) Photographs of xenograft tumors derived from transfected MDA-MB-231 cells. (B) Quantitative analysis of tumor volume. (C) Quantitative analysis of tumor weight. (D) The immunoreactivity of Ki-67 in xenograft tumors was analyzed by IHC. Scale bar, 50 μm. (E) Flow cytometry was performed to analyze the percentage of CD107a + NK cells in xenograft tumors. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.
    Figure Legend Snippet: Silencing of TYMSOS repressed tumor growth and boosted NK cell sensitivity in vivo (A) Photographs of xenograft tumors derived from transfected MDA-MB-231 cells. (B) Quantitative analysis of tumor volume. (C) Quantitative analysis of tumor weight. (D) The immunoreactivity of Ki-67 in xenograft tumors was analyzed by IHC. Scale bar, 50 μm. (E) Flow cytometry was performed to analyze the percentage of CD107a + NK cells in xenograft tumors. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.

    Techniques Used: In Vivo, Derivative Assay, Transfection, Flow Cytometry


    Figure Legend Snippet:

    Techniques Used: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, shRNA, Sequencing, Plasmid Preparation, Software, Confocal Microscopy

    human nk cell line nk92 cells  (ATCC)


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    ATCC human nk cell line nk92 cells
    CBX3 was identified as a downstream effector to be involved in TYMSOS-regulated breast cancer progression (A and B) The mRNA level of CBX3 in breast tumors (n = 58) and their normal counterparts (n = 58) were detected by RT-qPCR. (C) Pearson correlation analysis between TYMSOS and CBX3 in breast tumors. (D) Data analyses of CBX3 expression in breast cancer based on UALCAN database ( https://ualcan.path.uab.edu/index.html ). (E) The expression of CBX3 in MCF10A and breast cancer cells (Hs578T, T47D, MCF-7, MDA-MB-231, and MDA-MB-468 cells) were detected by RT-qPCR and Western blot, respectively. (F) The protein level of CBX3 in CBX3-knockdown MDA-MB-231 or CBX3-overexpressing MCF-7 cells was detected by Western blot. (G) Cell proliferation of transfected breast cancer cells was assessed by CCK-8 assay. (H) The colony-forming ability of transfected breast cancer cells was monitored by colony formation assay. (I) Cell migration and invasion of transfected breast cancer cells were detected by Transwell assays. Scale bar, 100 μm. Activated <t>NK92</t> cells were co-cultured with transfected breast cancer cells. (J) Cytotoxicity of NK92 cells was detected by CytoTox96 cytotoxicity assay. (K) The secreted IFN-γ and TNF-α in cell culture supernatant of co-culture system were detected by ELISA assay. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.
    Human Nk Cell Line Nk92 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "LncRNA TYMSOS facilitates breast cancer metastasis and immune escape through downregulating ULBP3"

    Article Title: LncRNA TYMSOS facilitates breast cancer metastasis and immune escape through downregulating ULBP3

    Journal: iScience

    doi: 10.1016/j.isci.2023.107556

    CBX3 was identified as a downstream effector to be involved in TYMSOS-regulated breast cancer progression (A and B) The mRNA level of CBX3 in breast tumors (n = 58) and their normal counterparts (n = 58) were detected by RT-qPCR. (C) Pearson correlation analysis between TYMSOS and CBX3 in breast tumors. (D) Data analyses of CBX3 expression in breast cancer based on UALCAN database ( https://ualcan.path.uab.edu/index.html ). (E) The expression of CBX3 in MCF10A and breast cancer cells (Hs578T, T47D, MCF-7, MDA-MB-231, and MDA-MB-468 cells) were detected by RT-qPCR and Western blot, respectively. (F) The protein level of CBX3 in CBX3-knockdown MDA-MB-231 or CBX3-overexpressing MCF-7 cells was detected by Western blot. (G) Cell proliferation of transfected breast cancer cells was assessed by CCK-8 assay. (H) The colony-forming ability of transfected breast cancer cells was monitored by colony formation assay. (I) Cell migration and invasion of transfected breast cancer cells were detected by Transwell assays. Scale bar, 100 μm. Activated NK92 cells were co-cultured with transfected breast cancer cells. (J) Cytotoxicity of NK92 cells was detected by CytoTox96 cytotoxicity assay. (K) The secreted IFN-γ and TNF-α in cell culture supernatant of co-culture system were detected by ELISA assay. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.
    Figure Legend Snippet: CBX3 was identified as a downstream effector to be involved in TYMSOS-regulated breast cancer progression (A and B) The mRNA level of CBX3 in breast tumors (n = 58) and their normal counterparts (n = 58) were detected by RT-qPCR. (C) Pearson correlation analysis between TYMSOS and CBX3 in breast tumors. (D) Data analyses of CBX3 expression in breast cancer based on UALCAN database ( https://ualcan.path.uab.edu/index.html ). (E) The expression of CBX3 in MCF10A and breast cancer cells (Hs578T, T47D, MCF-7, MDA-MB-231, and MDA-MB-468 cells) were detected by RT-qPCR and Western blot, respectively. (F) The protein level of CBX3 in CBX3-knockdown MDA-MB-231 or CBX3-overexpressing MCF-7 cells was detected by Western blot. (G) Cell proliferation of transfected breast cancer cells was assessed by CCK-8 assay. (H) The colony-forming ability of transfected breast cancer cells was monitored by colony formation assay. (I) Cell migration and invasion of transfected breast cancer cells were detected by Transwell assays. Scale bar, 100 μm. Activated NK92 cells were co-cultured with transfected breast cancer cells. (J) Cytotoxicity of NK92 cells was detected by CytoTox96 cytotoxicity assay. (K) The secreted IFN-γ and TNF-α in cell culture supernatant of co-culture system were detected by ELISA assay. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.

    Techniques Used: Quantitative RT-PCR, Expressing, Western Blot, Transfection, CCK-8 Assay, Colony Assay, Migration, Cell Culture, Cytotoxicity Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

    TYMSOS promoted the malignant phenotypes of breast cancer cells (A) The TYMSOS level in MCF10A and breast cancer cells (Hs578T, T47D, MCF-7, MDA-MB-231, and MDA-MB-468 cells) was detected by RT-qPCR. (B) The TYMSOS level in TYMSOS-knockdown MDA-MB-231 or TYMSOS-overexpressing MCF-7 cells were detected by RT-qPCR. (C) Cell proliferation of transfected breast cancer cells was assessed by CCK-8 assay. (D) The colony-forming ability of transfected breast cancer cells was monitored by colony formation assay. (E) Cell migration and invasion of transfected breast cancer cells were detected by Transwell assays. Scale bar, 100 μm. Activated NK92 cells were co-cultured with transfected breast cancer cells. (F) Cytotoxicity of NK92 cells was detected by CytoTox96 cytotoxicity assay. (G) The secreted IFN-γ and TNF-α in cell culture supernatant of co-culture system were detected by ELISA assay. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.
    Figure Legend Snippet: TYMSOS promoted the malignant phenotypes of breast cancer cells (A) The TYMSOS level in MCF10A and breast cancer cells (Hs578T, T47D, MCF-7, MDA-MB-231, and MDA-MB-468 cells) was detected by RT-qPCR. (B) The TYMSOS level in TYMSOS-knockdown MDA-MB-231 or TYMSOS-overexpressing MCF-7 cells were detected by RT-qPCR. (C) Cell proliferation of transfected breast cancer cells was assessed by CCK-8 assay. (D) The colony-forming ability of transfected breast cancer cells was monitored by colony formation assay. (E) Cell migration and invasion of transfected breast cancer cells were detected by Transwell assays. Scale bar, 100 μm. Activated NK92 cells were co-cultured with transfected breast cancer cells. (F) Cytotoxicity of NK92 cells was detected by CytoTox96 cytotoxicity assay. (G) The secreted IFN-γ and TNF-α in cell culture supernatant of co-culture system were detected by ELISA assay. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.

    Techniques Used: Quantitative RT-PCR, Transfection, CCK-8 Assay, Colony Assay, Migration, Cell Culture, Cytotoxicity Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

    Silencing of TYMSOS repressed tumor growth and boosted NK cell sensitivity in vivo (A) Photographs of xenograft tumors derived from transfected MDA-MB-231 cells. (B) Quantitative analysis of tumor volume. (C) Quantitative analysis of tumor weight. (D) The immunoreactivity of Ki-67 in xenograft tumors was analyzed by IHC. Scale bar, 50 μm. (E) Flow cytometry was performed to analyze the percentage of CD107a + NK cells in xenograft tumors. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.
    Figure Legend Snippet: Silencing of TYMSOS repressed tumor growth and boosted NK cell sensitivity in vivo (A) Photographs of xenograft tumors derived from transfected MDA-MB-231 cells. (B) Quantitative analysis of tumor volume. (C) Quantitative analysis of tumor weight. (D) The immunoreactivity of Ki-67 in xenograft tumors was analyzed by IHC. Scale bar, 50 μm. (E) Flow cytometry was performed to analyze the percentage of CD107a + NK cells in xenograft tumors. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.

    Techniques Used: In Vivo, Derivative Assay, Transfection, Flow Cytometry


    Figure Legend Snippet:

    Techniques Used: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, shRNA, Sequencing, Plasmid Preparation, Software, Confocal Microscopy

    human nk cell line nk92  (ATCC)


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    ATCC human nk cell line nk92
    ANGPTL3 enhancements elevated <t>NK</t> <t>cell</t> cytotoxicity to ovarian cancer cells. (A, B) <t>NK</t> <t>cells</t> activated by IL-2, or not, were co-cultured with ANGPTL3-overexpressed ovarian cancer cells in a Transwell system, including SKOV3 (A) and ES-2 (B) cells. Then, the mRNA expression of CD69 was detected by qRT-PCR. n = 4. (C, D) The contents of TNF-α (C) and IFN-γ (D) were evaluated in supernatants from the co-culture of IL-2-treated NK cells and ovarian cancer cells. n = 3. (E, F) NK cell-mediated cytotoxicity was analyzed using an LDH cytotoxic assay kit. (G) Ovarian cancer cell apoptosis was analyzed by flow cytometry. The percentage of apoptotic cells was analyzed by calculating the total percentage of early apoptotic (Annexin V + /PI − ) and late necrotic (Annexin V + /PI + ) cells. n = 3. The differences among multiple groups were analyzed using ANOVA with SNK post-hoc test. * P < 0.05 vs. control groups. # P < 0.05 vs. IL-2 groups. IL-2, interleukin 2; TNF-α, tumor necrosis factor-alpha; IFN-γ, interferon gamma; ANGPTL3, Angiopoietin-like protein 3; E:T, effector-to-target.
    Human Nk Cell Line Nk92, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "ANGPTL3 affects the metastatic potential and the susceptibility of ovarian cancer cells to natural killer cell-mediated cytotoxicity"

    Article Title: ANGPTL3 affects the metastatic potential and the susceptibility of ovarian cancer cells to natural killer cell-mediated cytotoxicity

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2023.e18799

    ANGPTL3 enhancements elevated NK cell cytotoxicity to ovarian cancer cells. (A, B) NK cells activated by IL-2, or not, were co-cultured with ANGPTL3-overexpressed ovarian cancer cells in a Transwell system, including SKOV3 (A) and ES-2 (B) cells. Then, the mRNA expression of CD69 was detected by qRT-PCR. n = 4. (C, D) The contents of TNF-α (C) and IFN-γ (D) were evaluated in supernatants from the co-culture of IL-2-treated NK cells and ovarian cancer cells. n = 3. (E, F) NK cell-mediated cytotoxicity was analyzed using an LDH cytotoxic assay kit. (G) Ovarian cancer cell apoptosis was analyzed by flow cytometry. The percentage of apoptotic cells was analyzed by calculating the total percentage of early apoptotic (Annexin V + /PI − ) and late necrotic (Annexin V + /PI + ) cells. n = 3. The differences among multiple groups were analyzed using ANOVA with SNK post-hoc test. * P < 0.05 vs. control groups. # P < 0.05 vs. IL-2 groups. IL-2, interleukin 2; TNF-α, tumor necrosis factor-alpha; IFN-γ, interferon gamma; ANGPTL3, Angiopoietin-like protein 3; E:T, effector-to-target.
    Figure Legend Snippet: ANGPTL3 enhancements elevated NK cell cytotoxicity to ovarian cancer cells. (A, B) NK cells activated by IL-2, or not, were co-cultured with ANGPTL3-overexpressed ovarian cancer cells in a Transwell system, including SKOV3 (A) and ES-2 (B) cells. Then, the mRNA expression of CD69 was detected by qRT-PCR. n = 4. (C, D) The contents of TNF-α (C) and IFN-γ (D) were evaluated in supernatants from the co-culture of IL-2-treated NK cells and ovarian cancer cells. n = 3. (E, F) NK cell-mediated cytotoxicity was analyzed using an LDH cytotoxic assay kit. (G) Ovarian cancer cell apoptosis was analyzed by flow cytometry. The percentage of apoptotic cells was analyzed by calculating the total percentage of early apoptotic (Annexin V + /PI − ) and late necrotic (Annexin V + /PI + ) cells. n = 3. The differences among multiple groups were analyzed using ANOVA with SNK post-hoc test. * P < 0.05 vs. control groups. # P < 0.05 vs. IL-2 groups. IL-2, interleukin 2; TNF-α, tumor necrosis factor-alpha; IFN-γ, interferon gamma; ANGPTL3, Angiopoietin-like protein 3; E:T, effector-to-target.

    Techniques Used: Cell Culture, Expressing, Quantitative RT-PCR, Co-Culture Assay, Flow Cytometry

    Blockage of the JAK/STAT3 signaling suppressed the metastatic potential and NK cell cytotoxicity to ovarian cancer cells. (A–C) SKOV3 ovarian cancer cells were treated with JAK/STAT3 pathway inhibitor Stattic or Dimethyl sulfoxide (DMSO) (negative control). Then, cell viability (A), invasion (B) and EMT marker expression (C, D) were detected. (E–I) IL-2-treated NK cells were co-cultured with ovarian cancer cells treated with Stattic. Then, the expression of CD69 (E) and contents of IFN-γ (F) and TNF-α (G) were determined by qRT-PCR and ELISA, separately. The subsequent effects of JAK/STAT3 pathway suppression on the cytotoxicity (H) and apoptosis (I) of cancer cells were also investigated. * P < 0.05 vs. control groups. # P < 0.05 vs. IL-2 groups. n = 3. The differences among three or four groups were analyzed using ANOVA with SNK post-hoc test. TGF-β, transforming growth factor-β; IL-2, interleukin 2; TNF-α, tumor necrosis factor-alpha; IFN-γ, interferon gamma; DMSO, Dimethyl sulfoxide.
    Figure Legend Snippet: Blockage of the JAK/STAT3 signaling suppressed the metastatic potential and NK cell cytotoxicity to ovarian cancer cells. (A–C) SKOV3 ovarian cancer cells were treated with JAK/STAT3 pathway inhibitor Stattic or Dimethyl sulfoxide (DMSO) (negative control). Then, cell viability (A), invasion (B) and EMT marker expression (C, D) were detected. (E–I) IL-2-treated NK cells were co-cultured with ovarian cancer cells treated with Stattic. Then, the expression of CD69 (E) and contents of IFN-γ (F) and TNF-α (G) were determined by qRT-PCR and ELISA, separately. The subsequent effects of JAK/STAT3 pathway suppression on the cytotoxicity (H) and apoptosis (I) of cancer cells were also investigated. * P < 0.05 vs. control groups. # P < 0.05 vs. IL-2 groups. n = 3. The differences among three or four groups were analyzed using ANOVA with SNK post-hoc test. TGF-β, transforming growth factor-β; IL-2, interleukin 2; TNF-α, tumor necrosis factor-alpha; IFN-γ, interferon gamma; DMSO, Dimethyl sulfoxide.

    Techniques Used: Negative Control, Marker, Expressing, Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    human nk cell line nk92  (ATCC)


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    ATCC human nk cell line nk92
    Human Nk Cell Line Nk92, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human nk cell line nk92  (ATCC)


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    ATCC human nk cell line nk92
    (A) HCC cells pretreated with Lipotecan and/or radiation were cocultured with <t>NK92</t> cells. Cells were seeded and treated with different doses of radiation (2.5–10 Gy) following 1-h pretreatment with Lipotecan, or DMSO vehicle. Cells were then cocultured with NK92 cells for an additional 7 days. The surviving fraction was determined as described in Materials and Methods. All experiments were performed independently in triplicate. (B) Quantitative results of HCC cells treated with Lipotecan and/or radiation for 24 h were immunolabeled with anti-MICA or anti-MICB antibodies and fluorescent signals were detected by flow cytometry analysis (C) Huh 7 and (D) PLC 5 cells with the isotype controls shown as green histograms. ** p <0.01, *** p <0.001, and **** p <0.0001. Data are means ± SD from a representative experiment of at least triplicate.
    Human Nk Cell Line Nk92, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Topoisomerase I Inhibition Radiosensitizing Hepatocellular Carcinoma by RNF144A-mediated DNA-PKcs Ubiquitination and Natural Killer Cell Cytotoxicity"

    Article Title: Topoisomerase I Inhibition Radiosensitizing Hepatocellular Carcinoma by RNF144A-mediated DNA-PKcs Ubiquitination and Natural Killer Cell Cytotoxicity

    Journal: Journal of Clinical and Translational Hepatology

    doi: 10.14218/JCTH.2022.00271

    (A) HCC cells pretreated with Lipotecan and/or radiation were cocultured with NK92 cells. Cells were seeded and treated with different doses of radiation (2.5–10 Gy) following 1-h pretreatment with Lipotecan, or DMSO vehicle. Cells were then cocultured with NK92 cells for an additional 7 days. The surviving fraction was determined as described in Materials and Methods. All experiments were performed independently in triplicate. (B) Quantitative results of HCC cells treated with Lipotecan and/or radiation for 24 h were immunolabeled with anti-MICA or anti-MICB antibodies and fluorescent signals were detected by flow cytometry analysis (C) Huh 7 and (D) PLC 5 cells with the isotype controls shown as green histograms. ** p <0.01, *** p <0.001, and **** p <0.0001. Data are means ± SD from a representative experiment of at least triplicate.
    Figure Legend Snippet: (A) HCC cells pretreated with Lipotecan and/or radiation were cocultured with NK92 cells. Cells were seeded and treated with different doses of radiation (2.5–10 Gy) following 1-h pretreatment with Lipotecan, or DMSO vehicle. Cells were then cocultured with NK92 cells for an additional 7 days. The surviving fraction was determined as described in Materials and Methods. All experiments were performed independently in triplicate. (B) Quantitative results of HCC cells treated with Lipotecan and/or radiation for 24 h were immunolabeled with anti-MICA or anti-MICB antibodies and fluorescent signals were detected by flow cytometry analysis (C) Huh 7 and (D) PLC 5 cells with the isotype controls shown as green histograms. ** p <0.01, *** p <0.001, and **** p <0.0001. Data are means ± SD from a representative experiment of at least triplicate.

    Techniques Used: Immunolabeling, Flow Cytometry

    (A) Immunohistochemical (IHC) staining of MICA/B in cross-sections of orthotopic liver tumors in (1) sham treatment (vehicle), (2) intravenous Lipotecan, (3) RT, and (4) the combination of Lipotecan and RT. (B) Percentage of MICA/B expression from the representative cross-sections. (C) Diagram of NK cell-mediated cytotoxic and differential radiosensitizing effects after RT combined with topoisomerase 1 (TOP1) inhibition of RNF144A-mediated DNA-PKcs ubiquitination and DNA repair pathways in Huh7 and PLC5 cells. ** p <0.01, *** p <0.001, and **** p <0.0001. Data are means ± SD from a representative experiment of at least triplicate.
    Figure Legend Snippet: (A) Immunohistochemical (IHC) staining of MICA/B in cross-sections of orthotopic liver tumors in (1) sham treatment (vehicle), (2) intravenous Lipotecan, (3) RT, and (4) the combination of Lipotecan and RT. (B) Percentage of MICA/B expression from the representative cross-sections. (C) Diagram of NK cell-mediated cytotoxic and differential radiosensitizing effects after RT combined with topoisomerase 1 (TOP1) inhibition of RNF144A-mediated DNA-PKcs ubiquitination and DNA repair pathways in Huh7 and PLC5 cells. ** p <0.01, *** p <0.001, and **** p <0.0001. Data are means ± SD from a representative experiment of at least triplicate.

    Techniques Used: Immunohistochemical staining, Immunohistochemistry, Expressing, Inhibition

    human nk cell line nk92  (ATCC)


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    ATCC human nk cell line nk92
    Engineering and establishment of <t>α-Cot-NK92</t> cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Human Nk Cell Line Nk92, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets"

    Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2022.1089369

    Engineering and establishment of α-Cot-NK92 cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: Engineering and establishment of α-Cot-NK92 cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Techniques Used: Expressing, Cell Culture, Transduction, Flow Cytometry, Incubation

    The action of α-Cot-NK92 cells depends on the type of conjugator. (A) Expression levels of HER2 and EGFR in various tumor cell lines, including mammary gland adenocarcinoma (AU565), ovarian adenocarcinoma (SK-OV-3), and epidermoid carcinoma (A431), were confirmed through FACS. Histograms are representative of at least three independent experiments. (B) Cytotoxicity against HER2 + , EGFR + , or HER2 + and EGFR + tumor cells mediated by α-Cot-NK92 cells with or without α-HER2-Cot or ZEGFR-Cot. The α-Cot-NK92 cells were co-cultured with calcein-stained tumor cells with or without a conjugator for 4 h at E:T ratios of 5:1, 1:1, and 0.5:1. The intensity of fluorescence emitted from the lysed target cells was measured using a fluorescence plate reader (SpectraMax i3x). All cytotoxicity data are represented as the mean ± S.D. of triplicate experiments. The statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05 vs. vehicle; ns: not significant. (C, D) IFN-γ and TNF-α secretion and CD107a expression in α-Cot-NK92 cells cultured with or without HER2 + EGFR + AU565 (C) or HER2 − EGFR + A431 (D) cells and with/without the conjugator (α-HER2-Cot or ZEGFR-Cot). Secreted IFN-γ and TNF-α levels were measured via ELISA using the medium of α-Cot-NK92 cells co-cultured with target cells at a 1:1 E:T ratio for 12 h. All experiments were performed in triplicate wells for each condition and repeated at least two times. Average values are shown as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t -test. *** P < 0.001; ** P < 0.01; ns: not significant. CD107a expression in α-Cot-NK92 cells was evaluated through FACS analysis after co-culture with target cells at a 5:1 E:T ratio for 4 h. Each value represents the percentage of CD56 + CD107a + cells in flow cytometric density plots. (E) Expression level of HER2 and EGFR in pulmonary adenocarcinoma (A549) confirmed through FACS analysis. Histograms are representative of at least three independent experiments. (F) Kinetics of α-Cot-NK92 cell-mediated tumor cell lysis using the xCELLigence real time cell analysis system. NK92 or α-Cot-NK92 cells were co-cultured with unlabeled A549 cells with/without ZEGFR-Cot at a 5:1 E:T ratio and monitored over time. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: The action of α-Cot-NK92 cells depends on the type of conjugator. (A) Expression levels of HER2 and EGFR in various tumor cell lines, including mammary gland adenocarcinoma (AU565), ovarian adenocarcinoma (SK-OV-3), and epidermoid carcinoma (A431), were confirmed through FACS. Histograms are representative of at least three independent experiments. (B) Cytotoxicity against HER2 + , EGFR + , or HER2 + and EGFR + tumor cells mediated by α-Cot-NK92 cells with or without α-HER2-Cot or ZEGFR-Cot. The α-Cot-NK92 cells were co-cultured with calcein-stained tumor cells with or without a conjugator for 4 h at E:T ratios of 5:1, 1:1, and 0.5:1. The intensity of fluorescence emitted from the lysed target cells was measured using a fluorescence plate reader (SpectraMax i3x). All cytotoxicity data are represented as the mean ± S.D. of triplicate experiments. The statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05 vs. vehicle; ns: not significant. (C, D) IFN-γ and TNF-α secretion and CD107a expression in α-Cot-NK92 cells cultured with or without HER2 + EGFR + AU565 (C) or HER2 − EGFR + A431 (D) cells and with/without the conjugator (α-HER2-Cot or ZEGFR-Cot). Secreted IFN-γ and TNF-α levels were measured via ELISA using the medium of α-Cot-NK92 cells co-cultured with target cells at a 1:1 E:T ratio for 12 h. All experiments were performed in triplicate wells for each condition and repeated at least two times. Average values are shown as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t -test. *** P < 0.001; ** P < 0.01; ns: not significant. CD107a expression in α-Cot-NK92 cells was evaluated through FACS analysis after co-culture with target cells at a 5:1 E:T ratio for 4 h. Each value represents the percentage of CD56 + CD107a + cells in flow cytometric density plots. (E) Expression level of HER2 and EGFR in pulmonary adenocarcinoma (A549) confirmed through FACS analysis. Histograms are representative of at least three independent experiments. (F) Kinetics of α-Cot-NK92 cell-mediated tumor cell lysis using the xCELLigence real time cell analysis system. NK92 or α-Cot-NK92 cells were co-cultured with unlabeled A549 cells with/without ZEGFR-Cot at a 5:1 E:T ratio and monitored over time. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Techniques Used: Expressing, Cell Culture, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Lysis

    Combination with conjugator enhances recognition of multiple antigens by α-Cot-NK92 cells. (A) Additive cytotoxic effects of α-Cot-NK92 cells in multi-targeting through co-treatment with α-HER2-Cot and ZEGFR-Cot. α-Cot-NK92 cells were co-cultured with calcein-stained AU565 cells with/without α-HER2-Cot (50 or 500 μg/mL), ZEGFR-Cot (5 or 50 ng/mL), or both α-HER2-Cot and ZEGFR-Cot at a 1:1 E:T ratio for 4 h, or were co-cultured with calcein-stained A549-Red-Fluc cells with/without α-HER2-Cot (5 or 500 μg/mL) and/or ZEGFR-Cot (2.5 or 50 ng/mL) at a 5:1 E:T ratio for 4 h. (B) To mimic the heterogeneity of the recurrent tumor, the EGFR + HER2 − MDA-MB-231 and EGFR − HER2 + MDA-MB-453 cells were mixed. On day 1, one group was not treated with α-Cot-NK92 cells (none) and the other groups were co-cultured with α-Cot-NK92 cells with/without ZEGFR-Cot at a 1:1 E:T ratio for 4 h. After removing α-Cot-NK92 cells, the remaining target cells were cultured for 2 d and then co-cultured with the α-Cot-NK92 cells with ZEGFR-cot or α-HER2-cot. (C) Cytotoxicity of α-Cot-NK92 cells to tumor cells on day 1 and (D) after 2 d in a model mimicking a recurrent tumor. Population change was measured using a FACS. All cytotoxicity data are presented as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer. α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92.
    Figure Legend Snippet: Combination with conjugator enhances recognition of multiple antigens by α-Cot-NK92 cells. (A) Additive cytotoxic effects of α-Cot-NK92 cells in multi-targeting through co-treatment with α-HER2-Cot and ZEGFR-Cot. α-Cot-NK92 cells were co-cultured with calcein-stained AU565 cells with/without α-HER2-Cot (50 or 500 μg/mL), ZEGFR-Cot (5 or 50 ng/mL), or both α-HER2-Cot and ZEGFR-Cot at a 1:1 E:T ratio for 4 h, or were co-cultured with calcein-stained A549-Red-Fluc cells with/without α-HER2-Cot (5 or 500 μg/mL) and/or ZEGFR-Cot (2.5 or 50 ng/mL) at a 5:1 E:T ratio for 4 h. (B) To mimic the heterogeneity of the recurrent tumor, the EGFR + HER2 − MDA-MB-231 and EGFR − HER2 + MDA-MB-453 cells were mixed. On day 1, one group was not treated with α-Cot-NK92 cells (none) and the other groups were co-cultured with α-Cot-NK92 cells with/without ZEGFR-Cot at a 1:1 E:T ratio for 4 h. After removing α-Cot-NK92 cells, the remaining target cells were cultured for 2 d and then co-cultured with the α-Cot-NK92 cells with ZEGFR-cot or α-HER2-cot. (C) Cytotoxicity of α-Cot-NK92 cells to tumor cells on day 1 and (D) after 2 d in a model mimicking a recurrent tumor. Population change was measured using a FACS. All cytotoxicity data are presented as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer. α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92.

    Techniques Used: Cell Culture, Staining

    α-Cot-NK92 cells acts in a conjugator-dependent manner at the immune synapse (A) Images depicting the sequential steps in the interaction between α-Cot-NK92 and AU565 cells with or without conjugators (α-HER2-Cot and ZEGFR-Cot). The α-Cot-NK92 and AU565 (HER2 + EGFR + ) cells were labeled with LysoSensor Green and DDAO-SE, respectively, and co-cultured in a medium containing propidium iodide to assess the lytic dynamics of α-Cot-NK92 cells with or without conjugators. Images were acquired using a fluorescence microscope. The snapshots are provided in Supplementary Mov1. (B) Percentage of α-Cot-NK92 cells remaining at each step after 2, 4, or 6 h of co-incubation with AU565 cells with/without conjugators. (C) Duration of the process, and (D) ratio of α-Cot-NK92 cells that kill target cells for target-contacting cells. Data are shown as the mean (red line) ± S.D. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001. Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: α-Cot-NK92 cells acts in a conjugator-dependent manner at the immune synapse (A) Images depicting the sequential steps in the interaction between α-Cot-NK92 and AU565 cells with or without conjugators (α-HER2-Cot and ZEGFR-Cot). The α-Cot-NK92 and AU565 (HER2 + EGFR + ) cells were labeled with LysoSensor Green and DDAO-SE, respectively, and co-cultured in a medium containing propidium iodide to assess the lytic dynamics of α-Cot-NK92 cells with or without conjugators. Images were acquired using a fluorescence microscope. The snapshots are provided in Supplementary Mov1. (B) Percentage of α-Cot-NK92 cells remaining at each step after 2, 4, or 6 h of co-incubation with AU565 cells with/without conjugators. (C) Duration of the process, and (D) ratio of α-Cot-NK92 cells that kill target cells for target-contacting cells. Data are shown as the mean (red line) ± S.D. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001. Cot, cotinine; NK, natural killer.

    Techniques Used: Labeling, Cell Culture, Fluorescence, Microscopy, Incubation

    α-Cot-NK92 cells with a conjugator inhibit tumor growth in an in vivo lung cancer model. (A) The schedule of in vivo studies using luciferase-expressing A549-Red-Fluc cells in mouse metastasis model treated with α-Cot-NK92 cells with or without ZEGFR-Cot. (B) Representative in vivo bioluminescence imaging of each group treated with α-Cot-NK92 cells with or without ZEGFR-Cot and Taxol (10 mg/kg body weight) compared to levels in the untreated group (none) on day 57 after tumor cell injection. (C) The tumor burden of each group (n = 10) in in vivo lungs quantified as total flux (photon/s), which was monitored weekly for 57 d after the A549-Red-Fluc injection. (D, E) Representative BLI (D) and total quantitative flux (E) in ex vivo lungs of each group extracted on day 57 after in vivo BLI. *** P < 0.001 vs. none; * P < 0.05 vs. vehicle; Tukey’s multiple comparison test; n.s.: not significant. Bioluminescent images were acquired after an i.p. injection of D -luciferin (150 mg/kg body weight) and analyzed using the Living Image Software. Total flux data were plotted as mean ± SD. Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: α-Cot-NK92 cells with a conjugator inhibit tumor growth in an in vivo lung cancer model. (A) The schedule of in vivo studies using luciferase-expressing A549-Red-Fluc cells in mouse metastasis model treated with α-Cot-NK92 cells with or without ZEGFR-Cot. (B) Representative in vivo bioluminescence imaging of each group treated with α-Cot-NK92 cells with or without ZEGFR-Cot and Taxol (10 mg/kg body weight) compared to levels in the untreated group (none) on day 57 after tumor cell injection. (C) The tumor burden of each group (n = 10) in in vivo lungs quantified as total flux (photon/s), which was monitored weekly for 57 d after the A549-Red-Fluc injection. (D, E) Representative BLI (D) and total quantitative flux (E) in ex vivo lungs of each group extracted on day 57 after in vivo BLI. *** P < 0.001 vs. none; * P < 0.05 vs. vehicle; Tukey’s multiple comparison test; n.s.: not significant. Bioluminescent images were acquired after an i.p. injection of D -luciferin (150 mg/kg body weight) and analyzed using the Living Image Software. Total flux data were plotted as mean ± SD. Cot, cotinine; NK, natural killer.

    Techniques Used: In Vivo, Luciferase, Expressing, Imaging, Injection, Ex Vivo, Software

    Monoclonal α-Cot-NK92 cell lines have different killing potentials. (A, B) α-Cot-NK92 clones were established through single cell sorting using FACS in an expression level-dependent manner for CAR-Myc. Black triangles with a line indicate the expression level of CAR-Myc. The ability of α-Cot-NK92 clones to lyse AU565 cells was assessed using co-culture with α-HER2-Cot (A: 500 ng/mL) or ZEGFR-Cot (B: 50 ng/mL) for 4 h at E:T ratios of 5:1, 2:1, and 1:1. Ve: vehicle. (C, D) Comparison of cytolytic potency between low (L8) and medium (M2) CAR-expressing α-Cot-NK92 clones through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells in a dose-dependent manner of α-HER2-Cot (C: from a concentration of 500 to 62.5 ng/mL, upper panel) or ZEGFR-Cot (D: from a concentration of 12.5 to 1.56 ng/mL, lower panel). (E) Comparison of cytolytic potency between M2 clone and heterogeneous α-Cot-NK92 cells through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells with α-HER2-Cot (125 ng/mL) or ZEGFR-Cot (6.25 ng/mL). (F) Relative expression level (%) and mean fluorescence intensity (MFI) of HER2 and EGFR in breast cancer cells (AU565) and normal lung cells (WI-38). (G) Cytolytic activity of NK92, monoclonal α-Cot-NK92-L8, and α-Cot-NK92-M2 cells with ZEGFR-Cot or HER2-Cot against AU565 and WI-38 cells. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: Monoclonal α-Cot-NK92 cell lines have different killing potentials. (A, B) α-Cot-NK92 clones were established through single cell sorting using FACS in an expression level-dependent manner for CAR-Myc. Black triangles with a line indicate the expression level of CAR-Myc. The ability of α-Cot-NK92 clones to lyse AU565 cells was assessed using co-culture with α-HER2-Cot (A: 500 ng/mL) or ZEGFR-Cot (B: 50 ng/mL) for 4 h at E:T ratios of 5:1, 2:1, and 1:1. Ve: vehicle. (C, D) Comparison of cytolytic potency between low (L8) and medium (M2) CAR-expressing α-Cot-NK92 clones through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells in a dose-dependent manner of α-HER2-Cot (C: from a concentration of 500 to 62.5 ng/mL, upper panel) or ZEGFR-Cot (D: from a concentration of 12.5 to 1.56 ng/mL, lower panel). (E) Comparison of cytolytic potency between M2 clone and heterogeneous α-Cot-NK92 cells through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells with α-HER2-Cot (125 ng/mL) or ZEGFR-Cot (6.25 ng/mL). (F) Relative expression level (%) and mean fluorescence intensity (MFI) of HER2 and EGFR in breast cancer cells (AU565) and normal lung cells (WI-38). (G) Cytolytic activity of NK92, monoclonal α-Cot-NK92-L8, and α-Cot-NK92-M2 cells with ZEGFR-Cot or HER2-Cot against AU565 and WI-38 cells. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Techniques Used: Clone Assay, FACS, Expressing, Co-Culture Assay, Concentration Assay, Fluorescence, Activity Assay

    nk92 egfp cd16 pta 8836 cell lines  (ATCC)


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    ATCC nk92 egfp cd16 pta 8836 cell lines
    Neuroblastoma-derived sEVs modulate NK cell maturation in vivo and NK cell-mediated ADCC in vitro. (A) Schematic of NK cell subpopulations. Created with BioRender.com. (B) Representative flow cytometry plots of splenic NK cell subpopulations isolated from 9464D-GD2 tumor-bearing mice receiving the indicated treatments as described in . (C) Quantification of the percentage of immature splenic NK cell (CD27 +CD11b-; left panel) and mature NK cell (CD27- CD11b+; right panel) subpopulations in 9464D-GD2 tumor-bearing mice treated as described in . Mean±SEM, n=7 per group. Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. (D–G) Human (IMR32) or murine (9464D-GD2) neuroblastoma cells treated as indicated in the presence or absence of <t>NK92-CD16-EGFP</t> cells (NK) and monitored for viability utilizing cell impermeant nucleic acid stain YOYO-3 with the IncuCyte S3 Live-Cell Analysis System. The cell-by-cell analysis module was used to quantify viable tumor cells (YOYO3- EGFP-). (D) Kinetic analysis of the IMR32 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=6. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (E) Percentage of viable IMR32 cells at 24 hours, n=6. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. **p<0.01; ***p<0.001. (F) Kinetic analysis of the 9464D-GD2 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=4. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (G) Percentage of viable 9464D-GD2 cells at 24 hours, n=4. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. *p<0.05; **p<0.01; ***p<0.001. ADCC, antibody-dependent cell-mediated cytotoxicity; ANOVA, analysis of variance; DN, double negative; DP, double positive; sEV, small extracellular vesicle.
    Nk92 Egfp Cd16 Pta 8836 Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Small extracellular vesicles induce resistance to anti-GD2 immunotherapy unveiling tipifarnib as an adjunct to neuroblastoma immunotherapy"

    Article Title: Small extracellular vesicles induce resistance to anti-GD2 immunotherapy unveiling tipifarnib as an adjunct to neuroblastoma immunotherapy

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1136/jitc-2021-004399

    Neuroblastoma-derived sEVs modulate NK cell maturation in vivo and NK cell-mediated ADCC in vitro. (A) Schematic of NK cell subpopulations. Created with BioRender.com. (B) Representative flow cytometry plots of splenic NK cell subpopulations isolated from 9464D-GD2 tumor-bearing mice receiving the indicated treatments as described in . (C) Quantification of the percentage of immature splenic NK cell (CD27 +CD11b-; left panel) and mature NK cell (CD27- CD11b+; right panel) subpopulations in 9464D-GD2 tumor-bearing mice treated as described in . Mean±SEM, n=7 per group. Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. (D–G) Human (IMR32) or murine (9464D-GD2) neuroblastoma cells treated as indicated in the presence or absence of NK92-CD16-EGFP cells (NK) and monitored for viability utilizing cell impermeant nucleic acid stain YOYO-3 with the IncuCyte S3 Live-Cell Analysis System. The cell-by-cell analysis module was used to quantify viable tumor cells (YOYO3- EGFP-). (D) Kinetic analysis of the IMR32 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=6. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (E) Percentage of viable IMR32 cells at 24 hours, n=6. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. **p<0.01; ***p<0.001. (F) Kinetic analysis of the 9464D-GD2 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=4. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (G) Percentage of viable 9464D-GD2 cells at 24 hours, n=4. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. *p<0.05; **p<0.01; ***p<0.001. ADCC, antibody-dependent cell-mediated cytotoxicity; ANOVA, analysis of variance; DN, double negative; DP, double positive; sEV, small extracellular vesicle.
    Figure Legend Snippet: Neuroblastoma-derived sEVs modulate NK cell maturation in vivo and NK cell-mediated ADCC in vitro. (A) Schematic of NK cell subpopulations. Created with BioRender.com. (B) Representative flow cytometry plots of splenic NK cell subpopulations isolated from 9464D-GD2 tumor-bearing mice receiving the indicated treatments as described in . (C) Quantification of the percentage of immature splenic NK cell (CD27 +CD11b-; left panel) and mature NK cell (CD27- CD11b+; right panel) subpopulations in 9464D-GD2 tumor-bearing mice treated as described in . Mean±SEM, n=7 per group. Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. (D–G) Human (IMR32) or murine (9464D-GD2) neuroblastoma cells treated as indicated in the presence or absence of NK92-CD16-EGFP cells (NK) and monitored for viability utilizing cell impermeant nucleic acid stain YOYO-3 with the IncuCyte S3 Live-Cell Analysis System. The cell-by-cell analysis module was used to quantify viable tumor cells (YOYO3- EGFP-). (D) Kinetic analysis of the IMR32 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=6. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (E) Percentage of viable IMR32 cells at 24 hours, n=6. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. **p<0.01; ***p<0.001. (F) Kinetic analysis of the 9464D-GD2 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=4. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (G) Percentage of viable 9464D-GD2 cells at 24 hours, n=4. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. *p<0.05; **p<0.01; ***p<0.001. ADCC, antibody-dependent cell-mediated cytotoxicity; ANOVA, analysis of variance; DN, double negative; DP, double positive; sEV, small extracellular vesicle.

    Techniques Used: Derivative Assay, In Vivo, In Vitro, Flow Cytometry, Isolation, Staining, ADCC Assay

    human nk cell line  (ATCC)


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    ATCC human nk cell line
    Human Nk Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human nk cell line nk92
    Engineering and establishment of <t>α-Cot-NK92</t> cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Human Nk Cell Line Nk92, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human nk cell line nk92 mi
    Engineering and establishment of <t>α-Cot-NK92</t> cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Human Nk Cell Line Nk92 Mi, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human nk cell line nk92 cells
    CBX3 was identified as a downstream effector to be involved in TYMSOS-regulated breast cancer progression (A and B) The mRNA level of CBX3 in breast tumors (n = 58) and their normal counterparts (n = 58) were detected by RT-qPCR. (C) Pearson correlation analysis between TYMSOS and CBX3 in breast tumors. (D) Data analyses of CBX3 expression in breast cancer based on UALCAN database ( https://ualcan.path.uab.edu/index.html ). (E) The expression of CBX3 in MCF10A and breast cancer cells (Hs578T, T47D, MCF-7, MDA-MB-231, and MDA-MB-468 cells) were detected by RT-qPCR and Western blot, respectively. (F) The protein level of CBX3 in CBX3-knockdown MDA-MB-231 or CBX3-overexpressing MCF-7 cells was detected by Western blot. (G) Cell proliferation of transfected breast cancer cells was assessed by CCK-8 assay. (H) The colony-forming ability of transfected breast cancer cells was monitored by colony formation assay. (I) Cell migration and invasion of transfected breast cancer cells were detected by Transwell assays. Scale bar, 100 μm. Activated <t>NK92</t> cells were co-cultured with transfected breast cancer cells. (J) Cytotoxicity of NK92 cells was detected by CytoTox96 cytotoxicity assay. (K) The secreted IFN-γ and TNF-α in cell culture supernatant of co-culture system were detected by ELISA assay. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.
    Human Nk Cell Line Nk92 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC nk92 egfp cd16 pta 8836 cell lines
    Neuroblastoma-derived sEVs modulate NK cell maturation in vivo and NK cell-mediated ADCC in vitro. (A) Schematic of NK cell subpopulations. Created with BioRender.com. (B) Representative flow cytometry plots of splenic NK cell subpopulations isolated from 9464D-GD2 tumor-bearing mice receiving the indicated treatments as described in . (C) Quantification of the percentage of immature splenic NK cell (CD27 +CD11b-; left panel) and mature NK cell (CD27- CD11b+; right panel) subpopulations in 9464D-GD2 tumor-bearing mice treated as described in . Mean±SEM, n=7 per group. Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. (D–G) Human (IMR32) or murine (9464D-GD2) neuroblastoma cells treated as indicated in the presence or absence of <t>NK92-CD16-EGFP</t> cells (NK) and monitored for viability utilizing cell impermeant nucleic acid stain YOYO-3 with the IncuCyte S3 Live-Cell Analysis System. The cell-by-cell analysis module was used to quantify viable tumor cells (YOYO3- EGFP-). (D) Kinetic analysis of the IMR32 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=6. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (E) Percentage of viable IMR32 cells at 24 hours, n=6. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. **p<0.01; ***p<0.001. (F) Kinetic analysis of the 9464D-GD2 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=4. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (G) Percentage of viable 9464D-GD2 cells at 24 hours, n=4. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. *p<0.05; **p<0.01; ***p<0.001. ADCC, antibody-dependent cell-mediated cytotoxicity; ANOVA, analysis of variance; DN, double negative; DP, double positive; sEV, small extracellular vesicle.
    Nk92 Egfp Cd16 Pta 8836 Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human nk cell line
    Neuroblastoma-derived sEVs modulate NK cell maturation in vivo and NK cell-mediated ADCC in vitro. (A) Schematic of NK cell subpopulations. Created with BioRender.com. (B) Representative flow cytometry plots of splenic NK cell subpopulations isolated from 9464D-GD2 tumor-bearing mice receiving the indicated treatments as described in . (C) Quantification of the percentage of immature splenic NK cell (CD27 +CD11b-; left panel) and mature NK cell (CD27- CD11b+; right panel) subpopulations in 9464D-GD2 tumor-bearing mice treated as described in . Mean±SEM, n=7 per group. Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. (D–G) Human (IMR32) or murine (9464D-GD2) neuroblastoma cells treated as indicated in the presence or absence of <t>NK92-CD16-EGFP</t> cells (NK) and monitored for viability utilizing cell impermeant nucleic acid stain YOYO-3 with the IncuCyte S3 Live-Cell Analysis System. The cell-by-cell analysis module was used to quantify viable tumor cells (YOYO3- EGFP-). (D) Kinetic analysis of the IMR32 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=6. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (E) Percentage of viable IMR32 cells at 24 hours, n=6. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. **p<0.01; ***p<0.001. (F) Kinetic analysis of the 9464D-GD2 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=4. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (G) Percentage of viable 9464D-GD2 cells at 24 hours, n=4. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. *p<0.05; **p<0.01; ***p<0.001. ADCC, antibody-dependent cell-mediated cytotoxicity; ANOVA, analysis of variance; DN, double negative; DP, double positive; sEV, small extracellular vesicle.
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    Engineering and establishment of α-Cot-NK92 cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Journal: Frontiers in Immunology

    Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

    doi: 10.3389/fimmu.2022.1089369

    Figure Lengend Snippet: Engineering and establishment of α-Cot-NK92 cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Article Snippet: The human NK cell line NK92 (CRL-2407; ATCC) was maintained in the ATCC-recommended medium.

    Techniques: Expressing, Cell Culture, Transduction, Flow Cytometry, Incubation

    The action of α-Cot-NK92 cells depends on the type of conjugator. (A) Expression levels of HER2 and EGFR in various tumor cell lines, including mammary gland adenocarcinoma (AU565), ovarian adenocarcinoma (SK-OV-3), and epidermoid carcinoma (A431), were confirmed through FACS. Histograms are representative of at least three independent experiments. (B) Cytotoxicity against HER2 + , EGFR + , or HER2 + and EGFR + tumor cells mediated by α-Cot-NK92 cells with or without α-HER2-Cot or ZEGFR-Cot. The α-Cot-NK92 cells were co-cultured with calcein-stained tumor cells with or without a conjugator for 4 h at E:T ratios of 5:1, 1:1, and 0.5:1. The intensity of fluorescence emitted from the lysed target cells was measured using a fluorescence plate reader (SpectraMax i3x). All cytotoxicity data are represented as the mean ± S.D. of triplicate experiments. The statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05 vs. vehicle; ns: not significant. (C, D) IFN-γ and TNF-α secretion and CD107a expression in α-Cot-NK92 cells cultured with or without HER2 + EGFR + AU565 (C) or HER2 − EGFR + A431 (D) cells and with/without the conjugator (α-HER2-Cot or ZEGFR-Cot). Secreted IFN-γ and TNF-α levels were measured via ELISA using the medium of α-Cot-NK92 cells co-cultured with target cells at a 1:1 E:T ratio for 12 h. All experiments were performed in triplicate wells for each condition and repeated at least two times. Average values are shown as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t -test. *** P < 0.001; ** P < 0.01; ns: not significant. CD107a expression in α-Cot-NK92 cells was evaluated through FACS analysis after co-culture with target cells at a 5:1 E:T ratio for 4 h. Each value represents the percentage of CD56 + CD107a + cells in flow cytometric density plots. (E) Expression level of HER2 and EGFR in pulmonary adenocarcinoma (A549) confirmed through FACS analysis. Histograms are representative of at least three independent experiments. (F) Kinetics of α-Cot-NK92 cell-mediated tumor cell lysis using the xCELLigence real time cell analysis system. NK92 or α-Cot-NK92 cells were co-cultured with unlabeled A549 cells with/without ZEGFR-Cot at a 5:1 E:T ratio and monitored over time. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Journal: Frontiers in Immunology

    Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

    doi: 10.3389/fimmu.2022.1089369

    Figure Lengend Snippet: The action of α-Cot-NK92 cells depends on the type of conjugator. (A) Expression levels of HER2 and EGFR in various tumor cell lines, including mammary gland adenocarcinoma (AU565), ovarian adenocarcinoma (SK-OV-3), and epidermoid carcinoma (A431), were confirmed through FACS. Histograms are representative of at least three independent experiments. (B) Cytotoxicity against HER2 + , EGFR + , or HER2 + and EGFR + tumor cells mediated by α-Cot-NK92 cells with or without α-HER2-Cot or ZEGFR-Cot. The α-Cot-NK92 cells were co-cultured with calcein-stained tumor cells with or without a conjugator for 4 h at E:T ratios of 5:1, 1:1, and 0.5:1. The intensity of fluorescence emitted from the lysed target cells was measured using a fluorescence plate reader (SpectraMax i3x). All cytotoxicity data are represented as the mean ± S.D. of triplicate experiments. The statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05 vs. vehicle; ns: not significant. (C, D) IFN-γ and TNF-α secretion and CD107a expression in α-Cot-NK92 cells cultured with or without HER2 + EGFR + AU565 (C) or HER2 − EGFR + A431 (D) cells and with/without the conjugator (α-HER2-Cot or ZEGFR-Cot). Secreted IFN-γ and TNF-α levels were measured via ELISA using the medium of α-Cot-NK92 cells co-cultured with target cells at a 1:1 E:T ratio for 12 h. All experiments were performed in triplicate wells for each condition and repeated at least two times. Average values are shown as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t -test. *** P < 0.001; ** P < 0.01; ns: not significant. CD107a expression in α-Cot-NK92 cells was evaluated through FACS analysis after co-culture with target cells at a 5:1 E:T ratio for 4 h. Each value represents the percentage of CD56 + CD107a + cells in flow cytometric density plots. (E) Expression level of HER2 and EGFR in pulmonary adenocarcinoma (A549) confirmed through FACS analysis. Histograms are representative of at least three independent experiments. (F) Kinetics of α-Cot-NK92 cell-mediated tumor cell lysis using the xCELLigence real time cell analysis system. NK92 or α-Cot-NK92 cells were co-cultured with unlabeled A549 cells with/without ZEGFR-Cot at a 5:1 E:T ratio and monitored over time. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Article Snippet: The human NK cell line NK92 (CRL-2407; ATCC) was maintained in the ATCC-recommended medium.

    Techniques: Expressing, Cell Culture, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Lysis

    Combination with conjugator enhances recognition of multiple antigens by α-Cot-NK92 cells. (A) Additive cytotoxic effects of α-Cot-NK92 cells in multi-targeting through co-treatment with α-HER2-Cot and ZEGFR-Cot. α-Cot-NK92 cells were co-cultured with calcein-stained AU565 cells with/without α-HER2-Cot (50 or 500 μg/mL), ZEGFR-Cot (5 or 50 ng/mL), or both α-HER2-Cot and ZEGFR-Cot at a 1:1 E:T ratio for 4 h, or were co-cultured with calcein-stained A549-Red-Fluc cells with/without α-HER2-Cot (5 or 500 μg/mL) and/or ZEGFR-Cot (2.5 or 50 ng/mL) at a 5:1 E:T ratio for 4 h. (B) To mimic the heterogeneity of the recurrent tumor, the EGFR + HER2 − MDA-MB-231 and EGFR − HER2 + MDA-MB-453 cells were mixed. On day 1, one group was not treated with α-Cot-NK92 cells (none) and the other groups were co-cultured with α-Cot-NK92 cells with/without ZEGFR-Cot at a 1:1 E:T ratio for 4 h. After removing α-Cot-NK92 cells, the remaining target cells were cultured for 2 d and then co-cultured with the α-Cot-NK92 cells with ZEGFR-cot or α-HER2-cot. (C) Cytotoxicity of α-Cot-NK92 cells to tumor cells on day 1 and (D) after 2 d in a model mimicking a recurrent tumor. Population change was measured using a FACS. All cytotoxicity data are presented as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer. α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92.

    Journal: Frontiers in Immunology

    Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

    doi: 10.3389/fimmu.2022.1089369

    Figure Lengend Snippet: Combination with conjugator enhances recognition of multiple antigens by α-Cot-NK92 cells. (A) Additive cytotoxic effects of α-Cot-NK92 cells in multi-targeting through co-treatment with α-HER2-Cot and ZEGFR-Cot. α-Cot-NK92 cells were co-cultured with calcein-stained AU565 cells with/without α-HER2-Cot (50 or 500 μg/mL), ZEGFR-Cot (5 or 50 ng/mL), or both α-HER2-Cot and ZEGFR-Cot at a 1:1 E:T ratio for 4 h, or were co-cultured with calcein-stained A549-Red-Fluc cells with/without α-HER2-Cot (5 or 500 μg/mL) and/or ZEGFR-Cot (2.5 or 50 ng/mL) at a 5:1 E:T ratio for 4 h. (B) To mimic the heterogeneity of the recurrent tumor, the EGFR + HER2 − MDA-MB-231 and EGFR − HER2 + MDA-MB-453 cells were mixed. On day 1, one group was not treated with α-Cot-NK92 cells (none) and the other groups were co-cultured with α-Cot-NK92 cells with/without ZEGFR-Cot at a 1:1 E:T ratio for 4 h. After removing α-Cot-NK92 cells, the remaining target cells were cultured for 2 d and then co-cultured with the α-Cot-NK92 cells with ZEGFR-cot or α-HER2-cot. (C) Cytotoxicity of α-Cot-NK92 cells to tumor cells on day 1 and (D) after 2 d in a model mimicking a recurrent tumor. Population change was measured using a FACS. All cytotoxicity data are presented as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer. α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92.

    Article Snippet: The human NK cell line NK92 (CRL-2407; ATCC) was maintained in the ATCC-recommended medium.

    Techniques: Cell Culture, Staining

    α-Cot-NK92 cells acts in a conjugator-dependent manner at the immune synapse (A) Images depicting the sequential steps in the interaction between α-Cot-NK92 and AU565 cells with or without conjugators (α-HER2-Cot and ZEGFR-Cot). The α-Cot-NK92 and AU565 (HER2 + EGFR + ) cells were labeled with LysoSensor Green and DDAO-SE, respectively, and co-cultured in a medium containing propidium iodide to assess the lytic dynamics of α-Cot-NK92 cells with or without conjugators. Images were acquired using a fluorescence microscope. The snapshots are provided in Supplementary Mov1. (B) Percentage of α-Cot-NK92 cells remaining at each step after 2, 4, or 6 h of co-incubation with AU565 cells with/without conjugators. (C) Duration of the process, and (D) ratio of α-Cot-NK92 cells that kill target cells for target-contacting cells. Data are shown as the mean (red line) ± S.D. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001. Cot, cotinine; NK, natural killer.

    Journal: Frontiers in Immunology

    Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

    doi: 10.3389/fimmu.2022.1089369

    Figure Lengend Snippet: α-Cot-NK92 cells acts in a conjugator-dependent manner at the immune synapse (A) Images depicting the sequential steps in the interaction between α-Cot-NK92 and AU565 cells with or without conjugators (α-HER2-Cot and ZEGFR-Cot). The α-Cot-NK92 and AU565 (HER2 + EGFR + ) cells were labeled with LysoSensor Green and DDAO-SE, respectively, and co-cultured in a medium containing propidium iodide to assess the lytic dynamics of α-Cot-NK92 cells with or without conjugators. Images were acquired using a fluorescence microscope. The snapshots are provided in Supplementary Mov1. (B) Percentage of α-Cot-NK92 cells remaining at each step after 2, 4, or 6 h of co-incubation with AU565 cells with/without conjugators. (C) Duration of the process, and (D) ratio of α-Cot-NK92 cells that kill target cells for target-contacting cells. Data are shown as the mean (red line) ± S.D. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001. Cot, cotinine; NK, natural killer.

    Article Snippet: The human NK cell line NK92 (CRL-2407; ATCC) was maintained in the ATCC-recommended medium.

    Techniques: Labeling, Cell Culture, Fluorescence, Microscopy, Incubation

    α-Cot-NK92 cells with a conjugator inhibit tumor growth in an in vivo lung cancer model. (A) The schedule of in vivo studies using luciferase-expressing A549-Red-Fluc cells in mouse metastasis model treated with α-Cot-NK92 cells with or without ZEGFR-Cot. (B) Representative in vivo bioluminescence imaging of each group treated with α-Cot-NK92 cells with or without ZEGFR-Cot and Taxol (10 mg/kg body weight) compared to levels in the untreated group (none) on day 57 after tumor cell injection. (C) The tumor burden of each group (n = 10) in in vivo lungs quantified as total flux (photon/s), which was monitored weekly for 57 d after the A549-Red-Fluc injection. (D, E) Representative BLI (D) and total quantitative flux (E) in ex vivo lungs of each group extracted on day 57 after in vivo BLI. *** P < 0.001 vs. none; * P < 0.05 vs. vehicle; Tukey’s multiple comparison test; n.s.: not significant. Bioluminescent images were acquired after an i.p. injection of D -luciferin (150 mg/kg body weight) and analyzed using the Living Image Software. Total flux data were plotted as mean ± SD. Cot, cotinine; NK, natural killer.

    Journal: Frontiers in Immunology

    Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

    doi: 10.3389/fimmu.2022.1089369

    Figure Lengend Snippet: α-Cot-NK92 cells with a conjugator inhibit tumor growth in an in vivo lung cancer model. (A) The schedule of in vivo studies using luciferase-expressing A549-Red-Fluc cells in mouse metastasis model treated with α-Cot-NK92 cells with or without ZEGFR-Cot. (B) Representative in vivo bioluminescence imaging of each group treated with α-Cot-NK92 cells with or without ZEGFR-Cot and Taxol (10 mg/kg body weight) compared to levels in the untreated group (none) on day 57 after tumor cell injection. (C) The tumor burden of each group (n = 10) in in vivo lungs quantified as total flux (photon/s), which was monitored weekly for 57 d after the A549-Red-Fluc injection. (D, E) Representative BLI (D) and total quantitative flux (E) in ex vivo lungs of each group extracted on day 57 after in vivo BLI. *** P < 0.001 vs. none; * P < 0.05 vs. vehicle; Tukey’s multiple comparison test; n.s.: not significant. Bioluminescent images were acquired after an i.p. injection of D -luciferin (150 mg/kg body weight) and analyzed using the Living Image Software. Total flux data were plotted as mean ± SD. Cot, cotinine; NK, natural killer.

    Article Snippet: The human NK cell line NK92 (CRL-2407; ATCC) was maintained in the ATCC-recommended medium.

    Techniques: In Vivo, Luciferase, Expressing, Imaging, Injection, Ex Vivo, Software

    Monoclonal α-Cot-NK92 cell lines have different killing potentials. (A, B) α-Cot-NK92 clones were established through single cell sorting using FACS in an expression level-dependent manner for CAR-Myc. Black triangles with a line indicate the expression level of CAR-Myc. The ability of α-Cot-NK92 clones to lyse AU565 cells was assessed using co-culture with α-HER2-Cot (A: 500 ng/mL) or ZEGFR-Cot (B: 50 ng/mL) for 4 h at E:T ratios of 5:1, 2:1, and 1:1. Ve: vehicle. (C, D) Comparison of cytolytic potency between low (L8) and medium (M2) CAR-expressing α-Cot-NK92 clones through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells in a dose-dependent manner of α-HER2-Cot (C: from a concentration of 500 to 62.5 ng/mL, upper panel) or ZEGFR-Cot (D: from a concentration of 12.5 to 1.56 ng/mL, lower panel). (E) Comparison of cytolytic potency between M2 clone and heterogeneous α-Cot-NK92 cells through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells with α-HER2-Cot (125 ng/mL) or ZEGFR-Cot (6.25 ng/mL). (F) Relative expression level (%) and mean fluorescence intensity (MFI) of HER2 and EGFR in breast cancer cells (AU565) and normal lung cells (WI-38). (G) Cytolytic activity of NK92, monoclonal α-Cot-NK92-L8, and α-Cot-NK92-M2 cells with ZEGFR-Cot or HER2-Cot against AU565 and WI-38 cells. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Journal: Frontiers in Immunology

    Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

    doi: 10.3389/fimmu.2022.1089369

    Figure Lengend Snippet: Monoclonal α-Cot-NK92 cell lines have different killing potentials. (A, B) α-Cot-NK92 clones were established through single cell sorting using FACS in an expression level-dependent manner for CAR-Myc. Black triangles with a line indicate the expression level of CAR-Myc. The ability of α-Cot-NK92 clones to lyse AU565 cells was assessed using co-culture with α-HER2-Cot (A: 500 ng/mL) or ZEGFR-Cot (B: 50 ng/mL) for 4 h at E:T ratios of 5:1, 2:1, and 1:1. Ve: vehicle. (C, D) Comparison of cytolytic potency between low (L8) and medium (M2) CAR-expressing α-Cot-NK92 clones through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells in a dose-dependent manner of α-HER2-Cot (C: from a concentration of 500 to 62.5 ng/mL, upper panel) or ZEGFR-Cot (D: from a concentration of 12.5 to 1.56 ng/mL, lower panel). (E) Comparison of cytolytic potency between M2 clone and heterogeneous α-Cot-NK92 cells through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells with α-HER2-Cot (125 ng/mL) or ZEGFR-Cot (6.25 ng/mL). (F) Relative expression level (%) and mean fluorescence intensity (MFI) of HER2 and EGFR in breast cancer cells (AU565) and normal lung cells (WI-38). (G) Cytolytic activity of NK92, monoclonal α-Cot-NK92-L8, and α-Cot-NK92-M2 cells with ZEGFR-Cot or HER2-Cot against AU565 and WI-38 cells. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Article Snippet: The human NK cell line NK92 (CRL-2407; ATCC) was maintained in the ATCC-recommended medium.

    Techniques: Clone Assay, FACS, Expressing, Co-Culture Assay, Concentration Assay, Fluorescence, Activity Assay

    CBX3 was identified as a downstream effector to be involved in TYMSOS-regulated breast cancer progression (A and B) The mRNA level of CBX3 in breast tumors (n = 58) and their normal counterparts (n = 58) were detected by RT-qPCR. (C) Pearson correlation analysis between TYMSOS and CBX3 in breast tumors. (D) Data analyses of CBX3 expression in breast cancer based on UALCAN database ( https://ualcan.path.uab.edu/index.html ). (E) The expression of CBX3 in MCF10A and breast cancer cells (Hs578T, T47D, MCF-7, MDA-MB-231, and MDA-MB-468 cells) were detected by RT-qPCR and Western blot, respectively. (F) The protein level of CBX3 in CBX3-knockdown MDA-MB-231 or CBX3-overexpressing MCF-7 cells was detected by Western blot. (G) Cell proliferation of transfected breast cancer cells was assessed by CCK-8 assay. (H) The colony-forming ability of transfected breast cancer cells was monitored by colony formation assay. (I) Cell migration and invasion of transfected breast cancer cells were detected by Transwell assays. Scale bar, 100 μm. Activated NK92 cells were co-cultured with transfected breast cancer cells. (J) Cytotoxicity of NK92 cells was detected by CytoTox96 cytotoxicity assay. (K) The secreted IFN-γ and TNF-α in cell culture supernatant of co-culture system were detected by ELISA assay. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.

    Journal: iScience

    Article Title: LncRNA TYMSOS facilitates breast cancer metastasis and immune escape through downregulating ULBP3

    doi: 10.1016/j.isci.2023.107556

    Figure Lengend Snippet: CBX3 was identified as a downstream effector to be involved in TYMSOS-regulated breast cancer progression (A and B) The mRNA level of CBX3 in breast tumors (n = 58) and their normal counterparts (n = 58) were detected by RT-qPCR. (C) Pearson correlation analysis between TYMSOS and CBX3 in breast tumors. (D) Data analyses of CBX3 expression in breast cancer based on UALCAN database ( https://ualcan.path.uab.edu/index.html ). (E) The expression of CBX3 in MCF10A and breast cancer cells (Hs578T, T47D, MCF-7, MDA-MB-231, and MDA-MB-468 cells) were detected by RT-qPCR and Western blot, respectively. (F) The protein level of CBX3 in CBX3-knockdown MDA-MB-231 or CBX3-overexpressing MCF-7 cells was detected by Western blot. (G) Cell proliferation of transfected breast cancer cells was assessed by CCK-8 assay. (H) The colony-forming ability of transfected breast cancer cells was monitored by colony formation assay. (I) Cell migration and invasion of transfected breast cancer cells were detected by Transwell assays. Scale bar, 100 μm. Activated NK92 cells were co-cultured with transfected breast cancer cells. (J) Cytotoxicity of NK92 cells was detected by CytoTox96 cytotoxicity assay. (K) The secreted IFN-γ and TNF-α in cell culture supernatant of co-culture system were detected by ELISA assay. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.

    Article Snippet: Human mammary epithelial cell line MCF10A and human breast cancer cell lines MCF-7, Hs578T, T47D, MDA-MB-231, MDA-MB-468 cells and human NK cell line NK92 cells were from ATCC (Manassas, VA, USA).

    Techniques: Quantitative RT-PCR, Expressing, Western Blot, Transfection, CCK-8 Assay, Colony Assay, Migration, Cell Culture, Cytotoxicity Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

    TYMSOS promoted the malignant phenotypes of breast cancer cells (A) The TYMSOS level in MCF10A and breast cancer cells (Hs578T, T47D, MCF-7, MDA-MB-231, and MDA-MB-468 cells) was detected by RT-qPCR. (B) The TYMSOS level in TYMSOS-knockdown MDA-MB-231 or TYMSOS-overexpressing MCF-7 cells were detected by RT-qPCR. (C) Cell proliferation of transfected breast cancer cells was assessed by CCK-8 assay. (D) The colony-forming ability of transfected breast cancer cells was monitored by colony formation assay. (E) Cell migration and invasion of transfected breast cancer cells were detected by Transwell assays. Scale bar, 100 μm. Activated NK92 cells were co-cultured with transfected breast cancer cells. (F) Cytotoxicity of NK92 cells was detected by CytoTox96 cytotoxicity assay. (G) The secreted IFN-γ and TNF-α in cell culture supernatant of co-culture system were detected by ELISA assay. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.

    Journal: iScience

    Article Title: LncRNA TYMSOS facilitates breast cancer metastasis and immune escape through downregulating ULBP3

    doi: 10.1016/j.isci.2023.107556

    Figure Lengend Snippet: TYMSOS promoted the malignant phenotypes of breast cancer cells (A) The TYMSOS level in MCF10A and breast cancer cells (Hs578T, T47D, MCF-7, MDA-MB-231, and MDA-MB-468 cells) was detected by RT-qPCR. (B) The TYMSOS level in TYMSOS-knockdown MDA-MB-231 or TYMSOS-overexpressing MCF-7 cells were detected by RT-qPCR. (C) Cell proliferation of transfected breast cancer cells was assessed by CCK-8 assay. (D) The colony-forming ability of transfected breast cancer cells was monitored by colony formation assay. (E) Cell migration and invasion of transfected breast cancer cells were detected by Transwell assays. Scale bar, 100 μm. Activated NK92 cells were co-cultured with transfected breast cancer cells. (F) Cytotoxicity of NK92 cells was detected by CytoTox96 cytotoxicity assay. (G) The secreted IFN-γ and TNF-α in cell culture supernatant of co-culture system were detected by ELISA assay. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.

    Article Snippet: Human mammary epithelial cell line MCF10A and human breast cancer cell lines MCF-7, Hs578T, T47D, MDA-MB-231, MDA-MB-468 cells and human NK cell line NK92 cells were from ATCC (Manassas, VA, USA).

    Techniques: Quantitative RT-PCR, Transfection, CCK-8 Assay, Colony Assay, Migration, Cell Culture, Cytotoxicity Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

    Silencing of TYMSOS repressed tumor growth and boosted NK cell sensitivity in vivo (A) Photographs of xenograft tumors derived from transfected MDA-MB-231 cells. (B) Quantitative analysis of tumor volume. (C) Quantitative analysis of tumor weight. (D) The immunoreactivity of Ki-67 in xenograft tumors was analyzed by IHC. Scale bar, 50 μm. (E) Flow cytometry was performed to analyze the percentage of CD107a + NK cells in xenograft tumors. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.

    Journal: iScience

    Article Title: LncRNA TYMSOS facilitates breast cancer metastasis and immune escape through downregulating ULBP3

    doi: 10.1016/j.isci.2023.107556

    Figure Lengend Snippet: Silencing of TYMSOS repressed tumor growth and boosted NK cell sensitivity in vivo (A) Photographs of xenograft tumors derived from transfected MDA-MB-231 cells. (B) Quantitative analysis of tumor volume. (C) Quantitative analysis of tumor weight. (D) The immunoreactivity of Ki-67 in xenograft tumors was analyzed by IHC. Scale bar, 50 μm. (E) Flow cytometry was performed to analyze the percentage of CD107a + NK cells in xenograft tumors. Data were analyzed from at least three independent experiments. Data were presented as mean ± S.D. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001 by Student’s t test or One-way ANOVA.

    Article Snippet: Human mammary epithelial cell line MCF10A and human breast cancer cell lines MCF-7, Hs578T, T47D, MDA-MB-231, MDA-MB-468 cells and human NK cell line NK92 cells were from ATCC (Manassas, VA, USA).

    Techniques: In Vivo, Derivative Assay, Transfection, Flow Cytometry

    Journal: iScience

    Article Title: LncRNA TYMSOS facilitates breast cancer metastasis and immune escape through downregulating ULBP3

    doi: 10.1016/j.isci.2023.107556

    Figure Lengend Snippet:

    Article Snippet: Human mammary epithelial cell line MCF10A and human breast cancer cell lines MCF-7, Hs578T, T47D, MDA-MB-231, MDA-MB-468 cells and human NK cell line NK92 cells were from ATCC (Manassas, VA, USA).

    Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, shRNA, Sequencing, Plasmid Preparation, Software, Confocal Microscopy

    Neuroblastoma-derived sEVs modulate NK cell maturation in vivo and NK cell-mediated ADCC in vitro. (A) Schematic of NK cell subpopulations. Created with BioRender.com. (B) Representative flow cytometry plots of splenic NK cell subpopulations isolated from 9464D-GD2 tumor-bearing mice receiving the indicated treatments as described in . (C) Quantification of the percentage of immature splenic NK cell (CD27 +CD11b-; left panel) and mature NK cell (CD27- CD11b+; right panel) subpopulations in 9464D-GD2 tumor-bearing mice treated as described in . Mean±SEM, n=7 per group. Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. (D–G) Human (IMR32) or murine (9464D-GD2) neuroblastoma cells treated as indicated in the presence or absence of NK92-CD16-EGFP cells (NK) and monitored for viability utilizing cell impermeant nucleic acid stain YOYO-3 with the IncuCyte S3 Live-Cell Analysis System. The cell-by-cell analysis module was used to quantify viable tumor cells (YOYO3- EGFP-). (D) Kinetic analysis of the IMR32 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=6. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (E) Percentage of viable IMR32 cells at 24 hours, n=6. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. **p<0.01; ***p<0.001. (F) Kinetic analysis of the 9464D-GD2 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=4. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (G) Percentage of viable 9464D-GD2 cells at 24 hours, n=4. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. *p<0.05; **p<0.01; ***p<0.001. ADCC, antibody-dependent cell-mediated cytotoxicity; ANOVA, analysis of variance; DN, double negative; DP, double positive; sEV, small extracellular vesicle.

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Small extracellular vesicles induce resistance to anti-GD2 immunotherapy unveiling tipifarnib as an adjunct to neuroblastoma immunotherapy

    doi: 10.1136/jitc-2021-004399

    Figure Lengend Snippet: Neuroblastoma-derived sEVs modulate NK cell maturation in vivo and NK cell-mediated ADCC in vitro. (A) Schematic of NK cell subpopulations. Created with BioRender.com. (B) Representative flow cytometry plots of splenic NK cell subpopulations isolated from 9464D-GD2 tumor-bearing mice receiving the indicated treatments as described in . (C) Quantification of the percentage of immature splenic NK cell (CD27 +CD11b-; left panel) and mature NK cell (CD27- CD11b+; right panel) subpopulations in 9464D-GD2 tumor-bearing mice treated as described in . Mean±SEM, n=7 per group. Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. (D–G) Human (IMR32) or murine (9464D-GD2) neuroblastoma cells treated as indicated in the presence or absence of NK92-CD16-EGFP cells (NK) and monitored for viability utilizing cell impermeant nucleic acid stain YOYO-3 with the IncuCyte S3 Live-Cell Analysis System. The cell-by-cell analysis module was used to quantify viable tumor cells (YOYO3- EGFP-). (D) Kinetic analysis of the IMR32 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=6. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (E) Percentage of viable IMR32 cells at 24 hours, n=6. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. **p<0.01; ***p<0.001. (F) Kinetic analysis of the 9464D-GD2 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=4. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (G) Percentage of viable 9464D-GD2 cells at 24 hours, n=4. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. *p<0.05; **p<0.01; ***p<0.001. ADCC, antibody-dependent cell-mediated cytotoxicity; ANOVA, analysis of variance; DN, double negative; DP, double positive; sEV, small extracellular vesicle.

    Article Snippet: Human IMR32 neuroblastoma (CCL-127), HEK 293T/17 (CRL-11268), and NK92-EGFP-CD16 (PTA-8836) cell lines were purchased from ATCC.

    Techniques: Derivative Assay, In Vivo, In Vitro, Flow Cytometry, Isolation, Staining, ADCC Assay