dmem  (thermo fisher)


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    Structured Review

    thermo fisher dmem
    Signaling molecules involved in ADM actions on PDX1– <t>β</t> -Lox5 cell aggregates. Aggregated β -Lox-5 cells transduced with PDX1 on day 7 of culture in <t>DMEM</t> containing 4 mg/mL glucose were treated with (A) KT5720 (KT, 10 µM), (B) PD98059 (PD, 10 µM), or (C) wortmannin (Wort, 0.1 µM) for 30 min and then ADM (0.1 µM) for 24 h, and insulin concentrations in the cell culture media were determined using human insulin ELISA kit. Data are displayed as mean ± SEM of the results from three separate experiments. Different letters indicate significant differences between groups ( P > 0.05).
    Dmem, supplied by thermo fisher, 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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    Images

    1) Product Images from "Circulating Adrenomedullin Is Elevated in Gestational Diabetes and Its Role in Impaired Insulin Production by β-Cells"

    Article Title: Circulating Adrenomedullin Is Elevated in Gestational Diabetes and Its Role in Impaired Insulin Production by β-Cells

    Journal: The Journal of Clinical Endocrinology and Metabolism

    doi: 10.1210/jc.2018-01119

    Signaling molecules involved in ADM actions on PDX1– β -Lox5 cell aggregates. Aggregated β -Lox-5 cells transduced with PDX1 on day 7 of culture in DMEM containing 4 mg/mL glucose were treated with (A) KT5720 (KT, 10 µM), (B) PD98059 (PD, 10 µM), or (C) wortmannin (Wort, 0.1 µM) for 30 min and then ADM (0.1 µM) for 24 h, and insulin concentrations in the cell culture media were determined using human insulin ELISA kit. Data are displayed as mean ± SEM of the results from three separate experiments. Different letters indicate significant differences between groups ( P > 0.05).
    Figure Legend Snippet: Signaling molecules involved in ADM actions on PDX1– β -Lox5 cell aggregates. Aggregated β -Lox-5 cells transduced with PDX1 on day 7 of culture in DMEM containing 4 mg/mL glucose were treated with (A) KT5720 (KT, 10 µM), (B) PD98059 (PD, 10 µM), or (C) wortmannin (Wort, 0.1 µM) for 30 min and then ADM (0.1 µM) for 24 h, and insulin concentrations in the cell culture media were determined using human insulin ELISA kit. Data are displayed as mean ± SEM of the results from three separate experiments. Different letters indicate significant differences between groups ( P > 0.05).

    Techniques Used: Transduction, Cell Culture, Enzyme-linked Immunosorbent Assay

    ADM inhibits insulin mRNA and secretion in PDX1– β -Lox5 cell aggregates. (A) mRNA for insulin in monolayer (Mono) or aggregated (Aggr) β -Lox-5 cells transduced with or without PDX1 on day 7 of culture in DMEM containing 4 mg/mL glucose was determined using real-time quantitative PCR and normalized to GAPDH mRNA expression. (B) Aggregated β -Lox-5 cells transduced with PDX1 on day 7 of culture in DMEM containing 4 mg/mL glucose were treated with ADM in the presence or absence of ADM22-52 for 24 h, and mRNA for insulin in the cells was determined using real-time quantitative PCR and normalized to GAPDH mRNA expression. (C) Insulin concentration in cell culture medium from (B) was determined using a human insulin ELISA kit. Data are displayed as mean ± SEM of the results from three separate experiments. Different letters indicate significant differences between groups ( P > 0.05).
    Figure Legend Snippet: ADM inhibits insulin mRNA and secretion in PDX1– β -Lox5 cell aggregates. (A) mRNA for insulin in monolayer (Mono) or aggregated (Aggr) β -Lox-5 cells transduced with or without PDX1 on day 7 of culture in DMEM containing 4 mg/mL glucose was determined using real-time quantitative PCR and normalized to GAPDH mRNA expression. (B) Aggregated β -Lox-5 cells transduced with PDX1 on day 7 of culture in DMEM containing 4 mg/mL glucose were treated with ADM in the presence or absence of ADM22-52 for 24 h, and mRNA for insulin in the cells was determined using real-time quantitative PCR and normalized to GAPDH mRNA expression. (C) Insulin concentration in cell culture medium from (B) was determined using a human insulin ELISA kit. Data are displayed as mean ± SEM of the results from three separate experiments. Different letters indicate significant differences between groups ( P > 0.05).

    Techniques Used: Transduction, Real-time Polymerase Chain Reaction, Expressing, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    Related Articles

    Concentration Assay:

    Article Title: Analysis of nonsense-mediated mRNA decay in mammalian cells.
    Article Snippet: .. The nonsense-mediated mRNA decay (NMD) pathway acts to selectively identify and degrade mRNAs that contain a premature translation termination codon (PTC), and hence reduce the accumulation of potentially toxic truncated proteins. ..

    Labeling:

    Article Title: Dynamic rewiring of the human interactome by interferon signaling
    Article Snippet: .. SILAC labeling and shotgun mass spectrometry HeLa cells were cultured in ­DMEM (Lys/Arg−/− ) supplemented with 10% dialyzed FBS (Invitrogen), 1× Pen-Strep, and combinations of the following lysine and arginine isotopologues: for “light” (“L”)-labeled cells, l -arginine (84 mg/L) and l -lysine (146 mg/L) (Sigma-Aldrich); for “medium” (“M”)-labeled cells, 13 C6 -l -arginine (87 mg/L) and D4 -l -lysine (150 mg/L); and for “heavy” (“H”)-labeled cells, 13 C6 15 N4 -l -arginine (89 mg/L) and 13 C6 15 N2 -l-lysine (154 mg/L) (Cambridge Isotope Laboratories). ..

    Mass Spectrometry:

    Article Title: Dynamic rewiring of the human interactome by interferon signaling
    Article Snippet: .. SILAC labeling and shotgun mass spectrometry HeLa cells were cultured in ­DMEM (Lys/Arg−/− ) supplemented with 10% dialyzed FBS (Invitrogen), 1× Pen-Strep, and combinations of the following lysine and arginine isotopologues: for “light” (“L”)-labeled cells, l -arginine (84 mg/L) and l -lysine (146 mg/L) (Sigma-Aldrich); for “medium” (“M”)-labeled cells, 13 C6 -l -arginine (87 mg/L) and D4 -l -lysine (150 mg/L); and for “heavy” (“H”)-labeled cells, 13 C6 15 N4 -l -arginine (89 mg/L) and 13 C6 15 N2 -l-lysine (154 mg/L) (Cambridge Isotope Laboratories). ..

    Cell Culture:

    Article Title: Dynamic rewiring of the human interactome by interferon signaling
    Article Snippet: .. SILAC labeling and shotgun mass spectrometry HeLa cells were cultured in ­DMEM (Lys/Arg−/− ) supplemented with 10% dialyzed FBS (Invitrogen), 1× Pen-Strep, and combinations of the following lysine and arginine isotopologues: for “light” (“L”)-labeled cells, l -arginine (84 mg/L) and l -lysine (146 mg/L) (Sigma-Aldrich); for “medium” (“M”)-labeled cells, 13 C6 -l -arginine (87 mg/L) and D4 -l -lysine (150 mg/L); and for “heavy” (“H”)-labeled cells, 13 C6 15 N4 -l -arginine (89 mg/L) and 13 C6 15 N2 -l-lysine (154 mg/L) (Cambridge Isotope Laboratories). ..

    other:

    Article Title: A primary cell model of HIV-1 latency that uses activation through the T cell receptor and return to quiescence to establish latent infection
    Article Snippet: For culturing 293T cells, supplement DMEM with 10% (vol/vol) HI FBS (Life Technologies) and 1% (vol/vol) penicillin-streptomycin.

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  • 99
    Thermo Fisher dmem f
    Reconstitution of signal-dependent retrieval of SSTR3 and GPR161. (A) Diagram of the signal-dependent retrieval systems under study. Left: Addition of sst triggers SSTR3 exit from cilia by directly activating SSTR3. Right: Addition of SAG activates the Hedgehog pathway and promotes GPR161 retrieval. SMO, Smoothened. (B) Kinetics of SSTR3 disappearance from cilia of primary hippocampal neurons and of IMCD3 stably expressing AP SSTR3 NG under the control of the TATA-less EF1α promoter were estimated by quantitation of immunofluorescence signals after addition of sst. The entire dataset for the sst condition is shown in Fig. S1 B. Data were fitted to a single exponential. Error bars indicate 95% confidence interval (CI). n = 280–424 cilia (neurons) and 57–80 cilia (IMCD3). (C) High-level expression of SSTR3 drives elongation of primary cilia. Top: AP SSTR3 GFP driven by various promoters or AP SSTR3 NG ) or an NG calibrator (see Materials and methods). Endogenous SSTR3 levels were estimated by comparative immunostaining (see Materials and methods). A Mann-Whitney test was used for pairwise comparisons of the number of SSTR3 molecules per cilia in neurons and in IMCD3 cells expressing AP SSTR3 NG or AP SSTR3 GFP under the control of pEF1αΔ. P > 0.05. n = 10–38 cilia. Error bars represent SD. Bottom: Effect of AP SSTR3 GFP expression on cilium length. Cilia lengths were measured in the GFP channel by live-cell imaging. n . (D) IMCD3-[pCrys- AP GPR161 NG3 ] were treated for 2 h with either SAG or vehicle. AP GPR161 NG3 was visualized by NG fluorescence, and basal bodies of cilia were stained with ninein. All cells were pretreated with the translation inhibitor emetine to eliminate signals from new protein synthesis. Bar, 4 µm. (E) Absolute quantitation of ciliary GPCR abundance. Top: Calibration of single-molecule fluorescence intensity. Bacterially expressed NG3 protein was spotted on glass coverslips (inset), and the fluorescent intensity of each individual NG3 was measured. n = 1,257 particles measured. Bottom: The three-step photobleaching of a representative spot shows that the fluorescence was emitted by a single NG3 molecule. The measured fluorescence intensity of NG3 was used to calibrate NG- and NG3-tagged SSTR3, GPR161, BBS5, and IFT88. Bar, 0.5 μm. (F) IMCD3-[pEF1αΔ- AP SSTR3 NG ] cells were treated with vehicle or sst for 2 h. Stable expression of an ER-localized biotin ligase BirA enables the biotinylation of AP SSTR3 with the biotin existing in the <t>DMEM/F-12</t> cell culture medium. Ciliary AP SSTR3 was pulse-labeled by Alexa Fluor 647–conjugated mSA (mSA647) for 5–10 min before imaging (see Materials and methods for details). Bar, 1 μm. The absolute number of AP SSTR3 NG molecules per cilia at t 0 was calculated by measuring the NG signal and using the NG3 calibrator. For all other time points, the ratio in ciliary mSA647 signal compared with t 0 was used to calculate the absolute number of molecules (see Materials and methods for details). Data were fitted to a single exponential. Error bars indicate 95% CI. n = 14 cilia. (G) IMCD3-[pCrys-GPR161 NG3 ] cells were treated with SAG or vehicle for 2 h. NG fluorescence was tracked in individual cilia, and the ratio of GPR161 NG3 to endogenous GPR161 was used to calculate the total levels of GPR161 as detailed in Materials and methods. Bar, 1 μm. Data were fitted to a single exponential. Error bars indicate 95% CI. n = 12–20 cilia.
    Dmem F, supplied by Thermo Fisher, 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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    99
    Thermo Fisher celltracker green cmfda dye
    V-ATPase inhibition by archazolid increases the adhesion of MDA-MB-231 cells onto HMEC-1 and the adhesion of PC-3 cells onto HUVECs. Confluent endothelial cells were treated with archazolid (arch) or DMSO (co) for 24 h. (A) Untreated MDA-MB-231 cells were stained with <t>CellTracker</t> Green <t>CMFDA</t> Dye and added to an HMEC-1 monolayer. The cells were allowed to adhere for 10 and 120 min. Non-adherent MDA-MB-231 cells were washed off. (B) Untreated PC-3 cells were stained with CellTracker Green CMFDA Dye and added to the HUVEC monolayer. The cells were allowed to adhere for 10, 30 and 60 min. Non-adherent PC-3 cells were washed off. (A, B) The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 3). *p ≤ 0.05 versus DMSO control.
    Celltracker Green Cmfda Dye, supplied by Thermo Fisher, 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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    Image Search Results


    Reconstitution of signal-dependent retrieval of SSTR3 and GPR161. (A) Diagram of the signal-dependent retrieval systems under study. Left: Addition of sst triggers SSTR3 exit from cilia by directly activating SSTR3. Right: Addition of SAG activates the Hedgehog pathway and promotes GPR161 retrieval. SMO, Smoothened. (B) Kinetics of SSTR3 disappearance from cilia of primary hippocampal neurons and of IMCD3 stably expressing AP SSTR3 NG under the control of the TATA-less EF1α promoter were estimated by quantitation of immunofluorescence signals after addition of sst. The entire dataset for the sst condition is shown in Fig. S1 B. Data were fitted to a single exponential. Error bars indicate 95% confidence interval (CI). n = 280–424 cilia (neurons) and 57–80 cilia (IMCD3). (C) High-level expression of SSTR3 drives elongation of primary cilia. Top: AP SSTR3 GFP driven by various promoters or AP SSTR3 NG ) or an NG calibrator (see Materials and methods). Endogenous SSTR3 levels were estimated by comparative immunostaining (see Materials and methods). A Mann-Whitney test was used for pairwise comparisons of the number of SSTR3 molecules per cilia in neurons and in IMCD3 cells expressing AP SSTR3 NG or AP SSTR3 GFP under the control of pEF1αΔ. P > 0.05. n = 10–38 cilia. Error bars represent SD. Bottom: Effect of AP SSTR3 GFP expression on cilium length. Cilia lengths were measured in the GFP channel by live-cell imaging. n . (D) IMCD3-[pCrys- AP GPR161 NG3 ] were treated for 2 h with either SAG or vehicle. AP GPR161 NG3 was visualized by NG fluorescence, and basal bodies of cilia were stained with ninein. All cells were pretreated with the translation inhibitor emetine to eliminate signals from new protein synthesis. Bar, 4 µm. (E) Absolute quantitation of ciliary GPCR abundance. Top: Calibration of single-molecule fluorescence intensity. Bacterially expressed NG3 protein was spotted on glass coverslips (inset), and the fluorescent intensity of each individual NG3 was measured. n = 1,257 particles measured. Bottom: The three-step photobleaching of a representative spot shows that the fluorescence was emitted by a single NG3 molecule. The measured fluorescence intensity of NG3 was used to calibrate NG- and NG3-tagged SSTR3, GPR161, BBS5, and IFT88. Bar, 0.5 μm. (F) IMCD3-[pEF1αΔ- AP SSTR3 NG ] cells were treated with vehicle or sst for 2 h. Stable expression of an ER-localized biotin ligase BirA enables the biotinylation of AP SSTR3 with the biotin existing in the DMEM/F-12 cell culture medium. Ciliary AP SSTR3 was pulse-labeled by Alexa Fluor 647–conjugated mSA (mSA647) for 5–10 min before imaging (see Materials and methods for details). Bar, 1 μm. The absolute number of AP SSTR3 NG molecules per cilia at t 0 was calculated by measuring the NG signal and using the NG3 calibrator. For all other time points, the ratio in ciliary mSA647 signal compared with t 0 was used to calculate the absolute number of molecules (see Materials and methods for details). Data were fitted to a single exponential. Error bars indicate 95% CI. n = 14 cilia. (G) IMCD3-[pCrys-GPR161 NG3 ] cells were treated with SAG or vehicle for 2 h. NG fluorescence was tracked in individual cilia, and the ratio of GPR161 NG3 to endogenous GPR161 was used to calculate the total levels of GPR161 as detailed in Materials and methods. Bar, 1 μm. Data were fitted to a single exponential. Error bars indicate 95% CI. n = 12–20 cilia.

    Journal: The Journal of Cell Biology

    Article Title: BBSome trains remove activated GPCRs from cilia by enabling passage through the transition zone

    doi: 10.1083/jcb.201709041

    Figure Lengend Snippet: Reconstitution of signal-dependent retrieval of SSTR3 and GPR161. (A) Diagram of the signal-dependent retrieval systems under study. Left: Addition of sst triggers SSTR3 exit from cilia by directly activating SSTR3. Right: Addition of SAG activates the Hedgehog pathway and promotes GPR161 retrieval. SMO, Smoothened. (B) Kinetics of SSTR3 disappearance from cilia of primary hippocampal neurons and of IMCD3 stably expressing AP SSTR3 NG under the control of the TATA-less EF1α promoter were estimated by quantitation of immunofluorescence signals after addition of sst. The entire dataset for the sst condition is shown in Fig. S1 B. Data were fitted to a single exponential. Error bars indicate 95% confidence interval (CI). n = 280–424 cilia (neurons) and 57–80 cilia (IMCD3). (C) High-level expression of SSTR3 drives elongation of primary cilia. Top: AP SSTR3 GFP driven by various promoters or AP SSTR3 NG ) or an NG calibrator (see Materials and methods). Endogenous SSTR3 levels were estimated by comparative immunostaining (see Materials and methods). A Mann-Whitney test was used for pairwise comparisons of the number of SSTR3 molecules per cilia in neurons and in IMCD3 cells expressing AP SSTR3 NG or AP SSTR3 GFP under the control of pEF1αΔ. P > 0.05. n = 10–38 cilia. Error bars represent SD. Bottom: Effect of AP SSTR3 GFP expression on cilium length. Cilia lengths were measured in the GFP channel by live-cell imaging. n . (D) IMCD3-[pCrys- AP GPR161 NG3 ] were treated for 2 h with either SAG or vehicle. AP GPR161 NG3 was visualized by NG fluorescence, and basal bodies of cilia were stained with ninein. All cells were pretreated with the translation inhibitor emetine to eliminate signals from new protein synthesis. Bar, 4 µm. (E) Absolute quantitation of ciliary GPCR abundance. Top: Calibration of single-molecule fluorescence intensity. Bacterially expressed NG3 protein was spotted on glass coverslips (inset), and the fluorescent intensity of each individual NG3 was measured. n = 1,257 particles measured. Bottom: The three-step photobleaching of a representative spot shows that the fluorescence was emitted by a single NG3 molecule. The measured fluorescence intensity of NG3 was used to calibrate NG- and NG3-tagged SSTR3, GPR161, BBS5, and IFT88. Bar, 0.5 μm. (F) IMCD3-[pEF1αΔ- AP SSTR3 NG ] cells were treated with vehicle or sst for 2 h. Stable expression of an ER-localized biotin ligase BirA enables the biotinylation of AP SSTR3 with the biotin existing in the DMEM/F-12 cell culture medium. Ciliary AP SSTR3 was pulse-labeled by Alexa Fluor 647–conjugated mSA (mSA647) for 5–10 min before imaging (see Materials and methods for details). Bar, 1 μm. The absolute number of AP SSTR3 NG molecules per cilia at t 0 was calculated by measuring the NG signal and using the NG3 calibrator. For all other time points, the ratio in ciliary mSA647 signal compared with t 0 was used to calculate the absolute number of molecules (see Materials and methods for details). Data were fitted to a single exponential. Error bars indicate 95% CI. n = 14 cilia. (G) IMCD3-[pCrys-GPR161 NG3 ] cells were treated with SAG or vehicle for 2 h. NG fluorescence was tracked in individual cilia, and the ratio of GPR161 NG3 to endogenous GPR161 was used to calculate the total levels of GPR161 as detailed in Materials and methods. Bar, 1 μm. Data were fitted to a single exponential. Error bars indicate 95% CI. n = 12–20 cilia.

    Article Snippet: Cells were imaged in DMEM/F-12 media with Hepes, no phenol red, and 0.2% FBS (11039-021; Gibco).

    Techniques: Stable Transfection, Expressing, Quantitation Assay, Immunofluorescence, Immunostaining, MANN-WHITNEY, Live Cell Imaging, Fluorescence, Staining, Cell Culture, Labeling, Imaging

    A NPC‐based phenotypic screening platform to discover inducers of OL differentiation and maturation. (a) Steps of OL differentiation from neurospheres generated from cortical NPCs of mouse E14.5 embryos: Neurosphere formation (NPC medium), OPC differentiation (OPC medium), and OL differentiation and maturation (OL medium). (b) Phase contrast (Top) and immunofluorescent staining of specific markers (Bottom) of NPCs (Nestin), OPCs (NG2), and OLs (MBP). Nuclei were stained with Hoechst. Scale bars, 40 μm. (c) The negative (DMSO) and positive (T3, 100 nM) controls of the high‐throughput screening system (Left). Scatter plot of the primary screen results of 7347 compounds (Right). The dotted line indicates two‐fold increase in the percentage of MBP + cells comparing to DMSO. (d) Representative images of MBP + ]

    Journal: Glia

    Article Title: Vitamin C promotes oligodendrocytes generation and remyelination. Vitamin C promotes oligodendrocytes generation and remyelination

    doi: 10.1002/glia.23306

    Figure Lengend Snippet: A NPC‐based phenotypic screening platform to discover inducers of OL differentiation and maturation. (a) Steps of OL differentiation from neurospheres generated from cortical NPCs of mouse E14.5 embryos: Neurosphere formation (NPC medium), OPC differentiation (OPC medium), and OL differentiation and maturation (OL medium). (b) Phase contrast (Top) and immunofluorescent staining of specific markers (Bottom) of NPCs (Nestin), OPCs (NG2), and OLs (MBP). Nuclei were stained with Hoechst. Scale bars, 40 μm. (c) The negative (DMSO) and positive (T3, 100 nM) controls of the high‐throughput screening system (Left). Scatter plot of the primary screen results of 7347 compounds (Right). The dotted line indicates two‐fold increase in the percentage of MBP + cells comparing to DMSO. (d) Representative images of MBP + ]

    Article Snippet: NPCs were expanded as neurospheres in the NPC medium (DMEM/F12 (Gibco), 20 ng mL−1 EGF, 20 ng mL−1 bFGF, 2% B27 (Invitrogen), 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin).

    Techniques: Generated, Staining, High Throughput Screening Assay

    Comparison of IVM for COCs cultured in the same media: DMEM-F12 (a), G1-PLUS (b), G2-PLUS (c), and TYH (d). The data were analyzed by the chi-squared test. A value of P

    Journal: Stem Cells International

    Article Title: Differentiation Potential of Human Wharton's Jelly-Derived Mesenchymal Stem Cells and Paracrine Signaling Interaction Contribute to Improve the In Vitro Maturation of Mouse Cumulus Oocyte Complexes

    doi: 10.1155/2018/7609284

    Figure Lengend Snippet: Comparison of IVM for COCs cultured in the same media: DMEM-F12 (a), G1-PLUS (b), G2-PLUS (c), and TYH (d). The data were analyzed by the chi-squared test. A value of P

    Article Snippet: The third experimental stage included the following types of coculture with hWJ-MSCs: (i) coculture in DMEM-F12, (j) coculture in G1-PLUS, (k) coculture in G2-PLUS, and (l) coculture in TYH.

    Techniques: Cell Culture

    qPCR analysis of OCT4 in hWJ-MSCs cultured under the 4 coculture conditions. In G1-PLUS, TYH, and G2-PLUS, OCT4 expression was downregulated to 25.9, 24.7, and 6.6%, respectively, compared to the OCT4 level in hWJ-MSCs cultured in DMEM-F12. The expression of OCT4 from hWJ-MSCs was not affected by the presence of COCs when cultured in DMEM-F12 (control group). The data was analyzed with the two-tailed paired Student's t -test. A P value

    Journal: Stem Cells International

    Article Title: Differentiation Potential of Human Wharton's Jelly-Derived Mesenchymal Stem Cells and Paracrine Signaling Interaction Contribute to Improve the In Vitro Maturation of Mouse Cumulus Oocyte Complexes

    doi: 10.1155/2018/7609284

    Figure Lengend Snippet: qPCR analysis of OCT4 in hWJ-MSCs cultured under the 4 coculture conditions. In G1-PLUS, TYH, and G2-PLUS, OCT4 expression was downregulated to 25.9, 24.7, and 6.6%, respectively, compared to the OCT4 level in hWJ-MSCs cultured in DMEM-F12. The expression of OCT4 from hWJ-MSCs was not affected by the presence of COCs when cultured in DMEM-F12 (control group). The data was analyzed with the two-tailed paired Student's t -test. A P value

    Article Snippet: The third experimental stage included the following types of coculture with hWJ-MSCs: (i) coculture in DMEM-F12, (j) coculture in G1-PLUS, (k) coculture in G2-PLUS, and (l) coculture in TYH.

    Techniques: Real-time Polymerase Chain Reaction, Cell Culture, Expressing, Two Tailed Test

    V-ATPase inhibition by archazolid increases the adhesion of MDA-MB-231 cells onto HMEC-1 and the adhesion of PC-3 cells onto HUVECs. Confluent endothelial cells were treated with archazolid (arch) or DMSO (co) for 24 h. (A) Untreated MDA-MB-231 cells were stained with CellTracker Green CMFDA Dye and added to an HMEC-1 monolayer. The cells were allowed to adhere for 10 and 120 min. Non-adherent MDA-MB-231 cells were washed off. (B) Untreated PC-3 cells were stained with CellTracker Green CMFDA Dye and added to the HUVEC monolayer. The cells were allowed to adhere for 10, 30 and 60 min. Non-adherent PC-3 cells were washed off. (A, B) The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 3). *p ≤ 0.05 versus DMSO control.

    Journal: PLoS ONE

    Article Title: The vacuolar-type ATPase inhibitor archazolid increases tumor cell adhesion to endothelial cells by accumulating extracellular collagen

    doi: 10.1371/journal.pone.0203053

    Figure Lengend Snippet: V-ATPase inhibition by archazolid increases the adhesion of MDA-MB-231 cells onto HMEC-1 and the adhesion of PC-3 cells onto HUVECs. Confluent endothelial cells were treated with archazolid (arch) or DMSO (co) for 24 h. (A) Untreated MDA-MB-231 cells were stained with CellTracker Green CMFDA Dye and added to an HMEC-1 monolayer. The cells were allowed to adhere for 10 and 120 min. Non-adherent MDA-MB-231 cells were washed off. (B) Untreated PC-3 cells were stained with CellTracker Green CMFDA Dye and added to the HUVEC monolayer. The cells were allowed to adhere for 10, 30 and 60 min. Non-adherent PC-3 cells were washed off. (A, B) The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 3). *p ≤ 0.05 versus DMSO control.

    Article Snippet: The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye (5 μM in serum-free DMEM, 37°C) for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37°C.

    Techniques: Inhibition, Multiple Displacement Amplification, Staining, Fluorescence

    Overexpression of cathepsin B in endothelial cells attenuates both the basal and the archazolid-induced tumor cell adhesion. HUVECs were transfected with a plasmid containing human cathepsin B (catB) or the empty vector (pcDNA3.1(-)delta MCS). After 48 h cells were treated with 1 nM archazolid (arch) or DMSO (co) for 24 h. (A) The expression of cathepsin B was determined by western blot analysis. One representative blot out of three independently performed experiments is shown. Actin served as loading control. (B) Untreated MDA-MB-231 cells were labeled with CellTracker Green CMFDA Dye, added to the HUVEC monolayer and were allowed to adhere for 10 min. Non-adherent MDA-MB-231 cells were washed off. The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 7). *p ≤ 0.05 versus DMSO (control transfection). # p ≤ 0.05 versus 1 nM archazolid (control transfection).

    Journal: PLoS ONE

    Article Title: The vacuolar-type ATPase inhibitor archazolid increases tumor cell adhesion to endothelial cells by accumulating extracellular collagen

    doi: 10.1371/journal.pone.0203053

    Figure Lengend Snippet: Overexpression of cathepsin B in endothelial cells attenuates both the basal and the archazolid-induced tumor cell adhesion. HUVECs were transfected with a plasmid containing human cathepsin B (catB) or the empty vector (pcDNA3.1(-)delta MCS). After 48 h cells were treated with 1 nM archazolid (arch) or DMSO (co) for 24 h. (A) The expression of cathepsin B was determined by western blot analysis. One representative blot out of three independently performed experiments is shown. Actin served as loading control. (B) Untreated MDA-MB-231 cells were labeled with CellTracker Green CMFDA Dye, added to the HUVEC monolayer and were allowed to adhere for 10 min. Non-adherent MDA-MB-231 cells were washed off. The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 7). *p ≤ 0.05 versus DMSO (control transfection). # p ≤ 0.05 versus 1 nM archazolid (control transfection).

    Article Snippet: The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye (5 μM in serum-free DMEM, 37°C) for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37°C.

    Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Western Blot, Multiple Displacement Amplification, Labeling, Fluorescence

    Blocking integrin β1 subunits on MDA-MB-231 or PC-3 cells prevents the archazolid-mediated increase in tumor cell adhesion. Confluent HUVECs were treated either with archazolid or DMSO for 24 h. MDA-MB-231 or PC-3 cells were stained with CellTracker Green CMFDA Dye. (A) The cell adhesion assay was performed after the integrin β1 subunit was blocked by a monoclonal antibody for 30 min on either MDA-MB-231 (n = 6) cells or HUVECs (n = 3). MDA-MB-231 cells were allowed to adhere for 10 min. (B) The cell adhesion assay was performed after the integrin β1 subunit was blocked by a monoclonal antibody for 30 min on either PC-3 cells (n = 5) or HUVECs (n = 3). PC-3 cells were allowed to adhere for 60 min. (A, B) Non-adherent tumor cells were washed off. The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM. *p ≤ 0.05 versus DMSO control, # p ≤ 0.05 versus 1 nM archazolid.

    Journal: PLoS ONE

    Article Title: The vacuolar-type ATPase inhibitor archazolid increases tumor cell adhesion to endothelial cells by accumulating extracellular collagen

    doi: 10.1371/journal.pone.0203053

    Figure Lengend Snippet: Blocking integrin β1 subunits on MDA-MB-231 or PC-3 cells prevents the archazolid-mediated increase in tumor cell adhesion. Confluent HUVECs were treated either with archazolid or DMSO for 24 h. MDA-MB-231 or PC-3 cells were stained with CellTracker Green CMFDA Dye. (A) The cell adhesion assay was performed after the integrin β1 subunit was blocked by a monoclonal antibody for 30 min on either MDA-MB-231 (n = 6) cells or HUVECs (n = 3). MDA-MB-231 cells were allowed to adhere for 10 min. (B) The cell adhesion assay was performed after the integrin β1 subunit was blocked by a monoclonal antibody for 30 min on either PC-3 cells (n = 5) or HUVECs (n = 3). PC-3 cells were allowed to adhere for 60 min. (A, B) Non-adherent tumor cells were washed off. The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM. *p ≤ 0.05 versus DMSO control, # p ≤ 0.05 versus 1 nM archazolid.

    Article Snippet: The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye (5 μM in serum-free DMEM, 37°C) for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37°C.

    Techniques: Blocking Assay, Multiple Displacement Amplification, Staining, Cell Adhesion Assay, Fluorescence

    V-ATPase inhibition by archazolid increases the adhesion of MDA-MB-231 cells onto HUVECs and decreases their transendothelial migration. (A, B) Confluent HUVECs were treated with archazolid (arch) or DMSO (co) for 24 h. Untreated MDA-MB-231 cells were stained with CellTracker Green CMFDA Dye and were added to the HUVEC monolayer. The cells were allowed to adhere for 10 min and 120 min. Non-adherent MDA-MB-231 cells were washed off. (A) The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 7). *p ≤ 0.05 versus DMSO control. (B) Microscopic images show unlabeled HUVECs and CellTracker Green CMFDA Dye-labeled MDA-MB-231 cells after 10 min of adhesion. Scale bar represents 200 μm. One representative image out of three independently performed experiments is shown. (C) HUVECs were grown on a porous filter membrane and were treated with archazolid for 24 h. Untreated CellTracker Green-labeled MDA-MB-231 cells were allowed to transmigrate through the endothelial monolayer for 24 h. Transmigrated cells were quantified by measuring the fluorescence signal. Data are expressed as mean ± SEM (n = 5). *p ≤ 0.05 versus FCS control.

    Journal: PLoS ONE

    Article Title: The vacuolar-type ATPase inhibitor archazolid increases tumor cell adhesion to endothelial cells by accumulating extracellular collagen

    doi: 10.1371/journal.pone.0203053

    Figure Lengend Snippet: V-ATPase inhibition by archazolid increases the adhesion of MDA-MB-231 cells onto HUVECs and decreases their transendothelial migration. (A, B) Confluent HUVECs were treated with archazolid (arch) or DMSO (co) for 24 h. Untreated MDA-MB-231 cells were stained with CellTracker Green CMFDA Dye and were added to the HUVEC monolayer. The cells were allowed to adhere for 10 min and 120 min. Non-adherent MDA-MB-231 cells were washed off. (A) The adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 7). *p ≤ 0.05 versus DMSO control. (B) Microscopic images show unlabeled HUVECs and CellTracker Green CMFDA Dye-labeled MDA-MB-231 cells after 10 min of adhesion. Scale bar represents 200 μm. One representative image out of three independently performed experiments is shown. (C) HUVECs were grown on a porous filter membrane and were treated with archazolid for 24 h. Untreated CellTracker Green-labeled MDA-MB-231 cells were allowed to transmigrate through the endothelial monolayer for 24 h. Transmigrated cells were quantified by measuring the fluorescence signal. Data are expressed as mean ± SEM (n = 5). *p ≤ 0.05 versus FCS control.

    Article Snippet: The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye (5 μM in serum-free DMEM, 37°C) for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37°C.

    Techniques: Inhibition, Multiple Displacement Amplification, Migration, Staining, Fluorescence, Labeling

    Collagen is the major ECM component mediating MDA-MB-231 and PC-3 cell adhesion. Archazolid increases the amount of extracellular collagen on HUVECs. (A) 24-well plates were coated with 10 μg/ml collagen (col), fibronectin (fn) or laminin (ln) or were left uncoated (co). Untreated MDA-MB-231 or PC-3 cells were stained with CellTracker Green CMFDA Dye and added into ECM-coated or uncoated wells. After 10 min of incubation, non-adherent cells were washed off. Adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 3). *p ≤ 0.05 versus uncoated control. (B) A cell adhesion assay onto collagen was performed with untreated MDA-MB-231 or PC-3 cells (co) and MDA-MB-231 or PC-3 cells on which the integrin β1 subunit was blocked by a monoclonal antibody (ab). Data are expressed as mean ± SEM (n = 3). * ,# p ≤ 0.05 versus control. (C) Confluent HUVECs were treated with 1 nM archazolid (arch) or DMSO (co) for 24 h. Surface collagen (green) was detected by immunofluorescence staining of viable cells and nuclei (blue) were visualized by Hoechst 33342 staining. Scale bar represents 20 μm. One representative image out of three independently performed experiments is shown. The increase in extracellular collagen was quantified. Data are expressed as mean ± SEM (n = 3). *p ≤ 0.05 versus DMSO control.

    Journal: PLoS ONE

    Article Title: The vacuolar-type ATPase inhibitor archazolid increases tumor cell adhesion to endothelial cells by accumulating extracellular collagen

    doi: 10.1371/journal.pone.0203053

    Figure Lengend Snippet: Collagen is the major ECM component mediating MDA-MB-231 and PC-3 cell adhesion. Archazolid increases the amount of extracellular collagen on HUVECs. (A) 24-well plates were coated with 10 μg/ml collagen (col), fibronectin (fn) or laminin (ln) or were left uncoated (co). Untreated MDA-MB-231 or PC-3 cells were stained with CellTracker Green CMFDA Dye and added into ECM-coated or uncoated wells. After 10 min of incubation, non-adherent cells were washed off. Adhesion was quantified by measuring the fluorescence signal using a plate reader. Data are expressed as mean ± SEM (n = 3). *p ≤ 0.05 versus uncoated control. (B) A cell adhesion assay onto collagen was performed with untreated MDA-MB-231 or PC-3 cells (co) and MDA-MB-231 or PC-3 cells on which the integrin β1 subunit was blocked by a monoclonal antibody (ab). Data are expressed as mean ± SEM (n = 3). * ,# p ≤ 0.05 versus control. (C) Confluent HUVECs were treated with 1 nM archazolid (arch) or DMSO (co) for 24 h. Surface collagen (green) was detected by immunofluorescence staining of viable cells and nuclei (blue) were visualized by Hoechst 33342 staining. Scale bar represents 20 μm. One representative image out of three independently performed experiments is shown. The increase in extracellular collagen was quantified. Data are expressed as mean ± SEM (n = 3). *p ≤ 0.05 versus DMSO control.

    Article Snippet: The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye (5 μM in serum-free DMEM, 37°C) for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37°C.

    Techniques: Multiple Displacement Amplification, Staining, Incubation, Fluorescence, Cell Adhesion Assay, Immunofluorescence