dulbecco s modified eagle s medium f12  (Thermo Fisher)


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    Name:
    DMEM F 12 no glutamine
    Description:
    Dulbecco s Modified Eagle Medium Nutrient Mixture F 12 DMEM F 12 is a widely used basal medium for supporting the growth of many different mammalian cells Cells successfully cultured in DMEM F 12 include MDCK glial cells fibroblasts human endothelial cells and rat fibroblasts We offer a variety of Gibco DMEM F 12 modifications for a range of cell culture applications Find the right formulation using the media selector tool This DMEM F 12 is modified as follows WithWithoutPhenol RedL glutamineHEPESThe complete formulation is available Gibco DMEM F 12 is a 1 1 mixture of DMEM and Ham s F 12 This formulation combines DMEM s high concentrations of glucose amino acids and vitamins with F 12 s wide variety of components Product UseFor in vitro diagnostic use CAUTION Not for human or animal therapeutic use Uses other than the intended use may be a violation of local law cGMP Manufacturing and Quality SystemGibco DMEM F 12 is manufactured at a cGMP compliant facility located in Paisley Scotland UK The facility is registered with the FDA as a medical device manufacturer and is certified to the ISO 13485 standard DMEM F 12 contains no proteins lipids or growth factors Therefore DMEM F 12 may require supplementation commonly with 10 Fetal Bovine Serum FBS DMEM F 12 uses a sodium bicarbonate buffer system and therefore requires a 5 10 CO2 environment to maintain physiological pH
    Catalog Number:
    21331020
    Price:
    None
    Category:
    Cell Culture Transfection Reagents
    Applications:
    Cell Culture|Mammalian Cell Culture
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    Structured Review

    Thermo Fisher dulbecco s modified eagle s medium f12
    Dulbecco s Modified Eagle Medium Nutrient Mixture F 12 DMEM F 12 is a widely used basal medium for supporting the growth of many different mammalian cells Cells successfully cultured in DMEM F 12 include MDCK glial cells fibroblasts human endothelial cells and rat fibroblasts We offer a variety of Gibco DMEM F 12 modifications for a range of cell culture applications Find the right formulation using the media selector tool This DMEM F 12 is modified as follows WithWithoutPhenol RedL glutamineHEPESThe complete formulation is available Gibco DMEM F 12 is a 1 1 mixture of DMEM and Ham s F 12 This formulation combines DMEM s high concentrations of glucose amino acids and vitamins with F 12 s wide variety of components Product UseFor in vitro diagnostic use CAUTION Not for human or animal therapeutic use Uses other than the intended use may be a violation of local law cGMP Manufacturing and Quality SystemGibco DMEM F 12 is manufactured at a cGMP compliant facility located in Paisley Scotland UK The facility is registered with the FDA as a medical device manufacturer and is certified to the ISO 13485 standard DMEM F 12 contains no proteins lipids or growth factors Therefore DMEM F 12 may require supplementation commonly with 10 Fetal Bovine Serum FBS DMEM F 12 uses a sodium bicarbonate buffer system and therefore requires a 5 10 CO2 environment to maintain physiological pH
    https://www.bioz.com/result/dulbecco s modified eagle s medium f12/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Related Articles

    Western Blot:

    Article Title: Nutrient-induced FNIP degradation by SCFβ-TRCP regulates FLCN complex localization and promotes renal cancer progression
    Article Snippet: .. Where indicated, HeLa, UOK-257, or UOK-257-2 cells were starved in Earle's Balanced Salt solution (Sigma-Aldrich) for 3 h and stimulated with 10% FBS, glucose-free or glutamine-free DMEM (Gibco) for a subsequent western blot or immunofluorescence analysis. .. Plasmids Flag-FNIP2 expression plasmid was described previously [ ].

    Immunofluorescence:

    Article Title: Nutrient-induced FNIP degradation by SCFβ-TRCP regulates FLCN complex localization and promotes renal cancer progression
    Article Snippet: .. Where indicated, HeLa, UOK-257, or UOK-257-2 cells were starved in Earle's Balanced Salt solution (Sigma-Aldrich) for 3 h and stimulated with 10% FBS, glucose-free or glutamine-free DMEM (Gibco) for a subsequent western blot or immunofluorescence analysis. .. Plasmids Flag-FNIP2 expression plasmid was described previously [ ].

    Staining:

    Article Title: Are serum-free and xeno-free culture conditions ideal for large scale clinical grade expansion of Wharton’s jelly derived mesenchymal stem cells? A comparative study
    Article Snippet: .. Annexin V staining We looked for the presence of apoptotic cells among cultures in DMEM KO + 10% FBS and MesenCult, using the Tali Apoptosis Kit – Annexin V Alexa Flour 488 and propidium iodide (PI; cat no. 10788; Invitrogen), according to the manufacturer’s instructions. .. After staining the cells with Annexin V Alexa Flour 488 and PI, the samples were loaded onto Tali Cellular Analysis Slides (cat no. T10794; Invitrogen), following which 20 images were captured using the Tali Image – Based Cytometer (cat no. T10796; Invitrogen).

    Mouse Assay:

    Article Title: Heat shock protein‐27 and sex‐selective regulation of muscarinic and proteinase‐activated receptor 2‐mediated vasodilatation: differential sensitivity to endothelial NOS inhibition
    Article Snippet: After removal of endotoxin with Detoxi‐gel columns (Fisher Scientific, Pittsburgh, PA, USA), the purity of the final rHSP27 and rC1 protein was more than 95% by SDS‐PAGE and the endotoxin concentration was < 5 EU·mg−1 protein (LAL assay). .. Aorta tissues harvested from female HSPB1‐null mice were cultured for either 3 or 24 h at 37°C in serum‐free DMEM containing 10 mM glucose (a euglycaemic value for mice), GlutaMAX™ supplement, with pyruvate (Life technologies, Canada, CAT #10567014), either in the absence or presence of 50 μg·mL−1 (2.2 μM) recombinant rHSP27 prepared as previously described (Salari et al., ). ..

    Cell Culture:

    Article Title: Heat shock protein‐27 and sex‐selective regulation of muscarinic and proteinase‐activated receptor 2‐mediated vasodilatation: differential sensitivity to endothelial NOS inhibition
    Article Snippet: After removal of endotoxin with Detoxi‐gel columns (Fisher Scientific, Pittsburgh, PA, USA), the purity of the final rHSP27 and rC1 protein was more than 95% by SDS‐PAGE and the endotoxin concentration was < 5 EU·mg−1 protein (LAL assay). .. Aorta tissues harvested from female HSPB1‐null mice were cultured for either 3 or 24 h at 37°C in serum‐free DMEM containing 10 mM glucose (a euglycaemic value for mice), GlutaMAX™ supplement, with pyruvate (Life technologies, Canada, CAT #10567014), either in the absence or presence of 50 μg·mL−1 (2.2 μM) recombinant rHSP27 prepared as previously described (Salari et al., ). ..

    Article Title: Senescence-associated secretory factors induced by cisplatin in melanoma cells promote non-senescent melanoma cell growth through activation of the ERK1/2-RSK1 pathway
    Article Snippet: At different intervals, alamarBlue was added and fluorescence intensity was detected using a microplate reader. .. A375 cells were seeded in triplicate and serum starved for 48 h. Then, DMEM medium with 0.25% FBS (negative control, NC), DMEM+ NS CM (1:1) and DMEM+ Sen CM (1:1) was added into one of the three wells, respectively, and cultured for 9 h. The cells were collected and mRNA array was performed using Affymetrix PrimeView Human Gene Expression Array (USA). ..

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Heat shock protein‐27 and sex‐selective regulation of muscarinic and proteinase‐activated receptor 2‐mediated vasodilatation: differential sensitivity to endothelial NOS inhibition
    Article Snippet: After removal of endotoxin with Detoxi‐gel columns (Fisher Scientific, Pittsburgh, PA, USA), the purity of the final rHSP27 and rC1 protein was more than 95% by SDS‐PAGE and the endotoxin concentration was < 5 EU·mg−1 protein (LAL assay). .. Aorta tissues harvested from female HSPB1‐null mice were cultured for either 3 or 24 h at 37°C in serum‐free DMEM containing 10 mM glucose (a euglycaemic value for mice), GlutaMAX™ supplement, with pyruvate (Life technologies, Canada, CAT #10567014), either in the absence or presence of 50 μg·mL−1 (2.2 μM) recombinant rHSP27 prepared as previously described (Salari et al., ). ..

    Recombinant:

    Article Title: Heat shock protein‐27 and sex‐selective regulation of muscarinic and proteinase‐activated receptor 2‐mediated vasodilatation: differential sensitivity to endothelial NOS inhibition
    Article Snippet: After removal of endotoxin with Detoxi‐gel columns (Fisher Scientific, Pittsburgh, PA, USA), the purity of the final rHSP27 and rC1 protein was more than 95% by SDS‐PAGE and the endotoxin concentration was < 5 EU·mg−1 protein (LAL assay). .. Aorta tissues harvested from female HSPB1‐null mice were cultured for either 3 or 24 h at 37°C in serum‐free DMEM containing 10 mM glucose (a euglycaemic value for mice), GlutaMAX™ supplement, with pyruvate (Life technologies, Canada, CAT #10567014), either in the absence or presence of 50 μg·mL−1 (2.2 μM) recombinant rHSP27 prepared as previously described (Salari et al., ). ..

    Migration:

    Article Title: Plexin-B2 Negatively Regulates Macrophage Motility, Rac, and Cdc42 Activation
    Article Snippet: .. Migration Macrophages were M-CSF starved in complete DMEM overnight, harvested, and seeded in plain DMEM at 2×105 per upper well of 96-well transwell plates with 8 µm pores (ChemoTx System; NeuroProbe, Gaithersburg, MD), over chemokines (Peprotech, Rocky Hill, NJ) in serum-free DMEM or FBS (Gibco, Carlsbad, California) in DMEM, and incubated at 37°C for 4 hours for migration towards chemokine or overnight for migration towards serum. .. Migrated cells were quantified using ToxiLight Bioassay Kit (Lonza, Rockland, ME).

    Incubation:

    Article Title: Plexin-B2 Negatively Regulates Macrophage Motility, Rac, and Cdc42 Activation
    Article Snippet: .. Migration Macrophages were M-CSF starved in complete DMEM overnight, harvested, and seeded in plain DMEM at 2×105 per upper well of 96-well transwell plates with 8 µm pores (ChemoTx System; NeuroProbe, Gaithersburg, MD), over chemokines (Peprotech, Rocky Hill, NJ) in serum-free DMEM or FBS (Gibco, Carlsbad, California) in DMEM, and incubated at 37°C for 4 hours for migration towards chemokine or overnight for migration towards serum. .. Migrated cells were quantified using ToxiLight Bioassay Kit (Lonza, Rockland, ME).

    Transfection:

    Article Title: Small-Molecule “BRCA1-Mimetics” Are Antagonists of Estrogen Receptor-α
    Article Snippet: Western blotting experiments showed that a minimum of 48 hours of exposure to 50 nM of BRCA1 -siRNA was required to obtain a large reduction ( > 75%) of the BRCA1 protein levels. .. Subconfluent cells in 24-well dishes were transfected overnight with 0.25 μg of each indicated vector plus the ERE-TK-Luc reporter in serum-free DMEM containing Lipofectamine 2000 (Life Technologies). ..

    Plasmid Preparation:

    Article Title: Small-Molecule “BRCA1-Mimetics” Are Antagonists of Estrogen Receptor-α
    Article Snippet: Western blotting experiments showed that a minimum of 48 hours of exposure to 50 nM of BRCA1 -siRNA was required to obtain a large reduction ( > 75%) of the BRCA1 protein levels. .. Subconfluent cells in 24-well dishes were transfected overnight with 0.25 μg of each indicated vector plus the ERE-TK-Luc reporter in serum-free DMEM containing Lipofectamine 2000 (Life Technologies). ..

    Negative Control:

    Article Title: Senescence-associated secretory factors induced by cisplatin in melanoma cells promote non-senescent melanoma cell growth through activation of the ERK1/2-RSK1 pathway
    Article Snippet: At different intervals, alamarBlue was added and fluorescence intensity was detected using a microplate reader. .. A375 cells were seeded in triplicate and serum starved for 48 h. Then, DMEM medium with 0.25% FBS (negative control, NC), DMEM+ NS CM (1:1) and DMEM+ Sen CM (1:1) was added into one of the three wells, respectively, and cultured for 9 h. The cells were collected and mRNA array was performed using Affymetrix PrimeView Human Gene Expression Array (USA). ..

    Expressing:

    Article Title: Senescence-associated secretory factors induced by cisplatin in melanoma cells promote non-senescent melanoma cell growth through activation of the ERK1/2-RSK1 pathway
    Article Snippet: At different intervals, alamarBlue was added and fluorescence intensity was detected using a microplate reader. .. A375 cells were seeded in triplicate and serum starved for 48 h. Then, DMEM medium with 0.25% FBS (negative control, NC), DMEM+ NS CM (1:1) and DMEM+ Sen CM (1:1) was added into one of the three wells, respectively, and cultured for 9 h. The cells were collected and mRNA array was performed using Affymetrix PrimeView Human Gene Expression Array (USA). ..

    Derivative Assay:

    Article Title: Developing a New Two-Step Protocol to Generate Functional Hepatocytes from Wharton's Jelly-Derived Mesenchymal Stem Cells under Hypoxic Condition
    Article Snippet: .. WJMSCs-SUT1 and WJMSCs-SUT2 were derived from the cultivation of WJ-MSCs in Dulbecco's modified Eagle's medium with 1.0 g/L glucose (DMEM-LG) (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA) and embryonic stem cells conditioned medium (ESCM) at 37°C with 5% CO2 and 5% O2 , respectively. ..

    Modification:

    Article Title: Developing a New Two-Step Protocol to Generate Functional Hepatocytes from Wharton's Jelly-Derived Mesenchymal Stem Cells under Hypoxic Condition
    Article Snippet: .. WJMSCs-SUT1 and WJMSCs-SUT2 were derived from the cultivation of WJ-MSCs in Dulbecco's modified Eagle's medium with 1.0 g/L glucose (DMEM-LG) (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA) and embryonic stem cells conditioned medium (ESCM) at 37°C with 5% CO2 and 5% O2 , respectively. ..

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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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    Average 99 stars, based on 1 article reviews
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    99
    Thermo Fisher dispase
    Post-detachment cell viability impacts the rate of cell expansion. (A) Comparison of cell numbers generated over 5 passages in mTeSR™1 on Matrigel™ using conventional colony scraping, Collagenase IV, <t>Dispase</t> or a 1 mM, 570 mOsmol/kg hypertonic sodium citrate solution to subculture the cells. Cell detachment methods were compared by continuously seeding 2×10 5 viable cells per well in six-well plates to control for differences in post-detachment cell recovery. When the hESC colonies for each condition reached confluence, cells were passaged and the viable number of cells determined. Three replicate wells were then individually re-plated at 2×10 5 and the process repeated. (B) The actual viable cell number determined at each passage was used to determine the total number of viable cells that would have been generated if all cells at each passage had been plated. Inset illustrates day 0 to day 15 with an expanded Y axis to illustrate the earlier passages. Error bars indicate standard error of the mean. All conditions, n = 3.
    Dispase, 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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    Average 99 stars, based on 1 article reviews
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    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

    Post-detachment cell viability impacts the rate of cell expansion. (A) Comparison of cell numbers generated over 5 passages in mTeSR™1 on Matrigel™ using conventional colony scraping, Collagenase IV, Dispase or a 1 mM, 570 mOsmol/kg hypertonic sodium citrate solution to subculture the cells. Cell detachment methods were compared by continuously seeding 2×10 5 viable cells per well in six-well plates to control for differences in post-detachment cell recovery. When the hESC colonies for each condition reached confluence, cells were passaged and the viable number of cells determined. Three replicate wells were then individually re-plated at 2×10 5 and the process repeated. (B) The actual viable cell number determined at each passage was used to determine the total number of viable cells that would have been generated if all cells at each passage had been plated. Inset illustrates day 0 to day 15 with an expanded Y axis to illustrate the earlier passages. Error bars indicate standard error of the mean. All conditions, n = 3.

    Journal: PLoS ONE

    Article Title: Scalable Passaging of Adherent Human Pluripotent Stem Cells

    doi: 10.1371/journal.pone.0088012

    Figure Lengend Snippet: Post-detachment cell viability impacts the rate of cell expansion. (A) Comparison of cell numbers generated over 5 passages in mTeSR™1 on Matrigel™ using conventional colony scraping, Collagenase IV, Dispase or a 1 mM, 570 mOsmol/kg hypertonic sodium citrate solution to subculture the cells. Cell detachment methods were compared by continuously seeding 2×10 5 viable cells per well in six-well plates to control for differences in post-detachment cell recovery. When the hESC colonies for each condition reached confluence, cells were passaged and the viable number of cells determined. Three replicate wells were then individually re-plated at 2×10 5 and the process repeated. (B) The actual viable cell number determined at each passage was used to determine the total number of viable cells that would have been generated if all cells at each passage had been plated. Inset illustrates day 0 to day 15 with an expanded Y axis to illustrate the earlier passages. Error bars indicate standard error of the mean. All conditions, n = 3.

    Article Snippet: Following removal of the medium, either 1 ml of Dispase (Life Technologies 17105-041, reconstituted at 1 mg/ml in DMEM/F12) or 1 ml of Collagenase Type IV (Life Technologies 17104-019, reconstituted at 1 mg/ml in DMEM/F12) was added to each well and allowed to incubate on the cells for 5 minutes at 37°C.

    Techniques: Generated