heat inactivated fbs  (GE Healthcare)

Journal: Frontiers in Aging Neuroscience

Article Title: Amyloid β-Induced Redistribution of Transcriptional Factor EB and Lysosomal Dysfunction in Primary Microglial Cells

doi: 10.3389/fnagi.2017.00228

Figure Lengend Snippet: Effects of Aβ on intracellular lysosomal pH value in primary microglia. Microglial cells were harvested and maintained in the 96-well plate, with DMEM-F12 containing 2% FBS at least for 2 h for the return of a resting state. After treated with the indicated concentration of Aβ for the indicated time, microglia cells were rinsed with live cell imaging solution. Intracellular pH of microglia was detected using fluorescent dye-based pHrodo Red AM intracellular pH indicator. The wavelength of 560 and 580 nm fluorescent light for excitation and emission was used in a Gemini EM microtiter plate reader. Forskolin (100 μM), a powerful protein kinase A (PKA) activator, capable of enhancing lysosomal acidification in intact cell, was served as control. Intracellular pH values were determined by the value of the arbitrary pH ratios of light excited at 560 nm and emitted at 580 nm with pH calibration curve kit (Invitrogen). Data were collected from 3 independent experiments with 8-paralleling wells. * P

Article Snippet: DMEM-F12 and heat-inactivated endotoxin-free fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT).

Techniques: Concentration Assay, Live Cell Imaging

Journal: PLoS ONE

Article Title: A novel function of N-linked glycoproteins, alpha-2-HS-glycoprotein and hemopexin: Implications for small molecule compound-mediated neuroprotection

doi: 10.1371/journal.pone.0186227

Figure Lengend Snippet: Protein factors in FBS exert WN1316-mediated cytoprotective activity. (A) Effect of FBS on WN1316-induced cytoprotection. Differentiated SH-SY5Y cells were treated with 8 μM WN1316 or DMSO for 3 h followed by 12 h of chase incubation without the compound, and then exposed to 40 μM menadione for 4 h. The cell viability was measured by AlamarBlue assay, and was expressed as a relative value (relative cytoprotective activity; -fold) of the WN1316-treated samples for vehicle control (DMSO) set as 1. Data are expressed as mean ± SD (n = 4). Statistical significance was evaluated by one-way ANOVA ( p

Article Snippet: Anti-oxidative stress-induced neuronal cell death (AOND) assay SH-SY5Y cells were grown in DMEM supplemented with 10% non-heat inactivated FBS (HyClone, Thermo Fisher Scientific, Waltham, MA), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in 5% CO2 .

Techniques: Activity Assay, Incubation, Alamar Blue Assay

Journal: PLoS ONE

Article Title: A novel function of N-linked glycoproteins, alpha-2-HS-glycoprotein and hemopexin: Implications for small molecule compound-mediated neuroprotection

doi: 10.1371/journal.pone.0186227

Figure Lengend Snippet: Deglycosylation does not affect WN1316-mediated cytoprotective activity. Differentiated SH-SY5Y cells were pretreated with 8 μM WN1316 in DMEM containing 0.28 mg/ml Blue pass treated with or without EnzMix and PNGaseF for 3 h followed by 12 h of chase incubation without the compound, and then exposed to 40 μM menadione for 4 h. As control experiments, differentiated SH-SY5Y cells were treated with 8 μM WN1316 (as a positive control) or DMSO (as a vehicle control) in DMEM supplemented with 2% FBS (corresponds to a protein concentration of approximately 0.5 mg/ml). The cell viability was calculated by AlamarBlue, and was expressed as a relative value (relative cytoprotective activity; -fold) of the WN1316-treated samples for vehicle control (DMSO) set as 1. Data are expressed as mean ± SD (n = 4). Statistical significance was evaluated by one-way ANOVA ( p

Article Snippet: Anti-oxidative stress-induced neuronal cell death (AOND) assay SH-SY5Y cells were grown in DMEM supplemented with 10% non-heat inactivated FBS (HyClone, Thermo Fisher Scientific, Waltham, MA), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in 5% CO2 .

Techniques: Activity Assay, Incubation, Positive Control, Protein Concentration

Journal: PLoS ONE

Article Title: A novel function of N-linked glycoproteins, alpha-2-HS-glycoprotein and hemopexin: Implications for small molecule compound-mediated neuroprotection

doi: 10.1371/journal.pone.0186227

Figure Lengend Snippet: N -linked glycoproteins exert WN1316-mediated cytoprotective activity. Differentiated SH-SY5Y cells were pretreated with 8 μM WN1316 in DMEM containing 0.2 mg/ml FBS, Con A pass, or Con A elute for 3 h followed by 12 h of chase incubation without the compound, and then exposed to 40 μM menadione for 4 h. As control experiments, differentiated SH-SY5Y cells were treated with 8 μM WN1316 (as a positive control) or DMSO (as a vehicle control) in DMEM supplemented with 2% FBS (corresponds to a protein concentration of approximately 0.5 mg/ml). The cell viability was calculated by AlamarBlue, and was expressed as a relative value (relative cytoprotective activity; -fold) of the WN1316-treated samples for vehicle control (DMSO) set as 1. Data are expressed as mean ± SD (n = 4). Statistical significance was evaluated by one-way ANOVA ( p

Article Snippet: Anti-oxidative stress-induced neuronal cell death (AOND) assay SH-SY5Y cells were grown in DMEM supplemented with 10% non-heat inactivated FBS (HyClone, Thermo Fisher Scientific, Waltham, MA), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in 5% CO2 .

Techniques: Activity Assay, Incubation, Positive Control, Protein Concentration

Journal: PLoS ONE

Article Title: A novel function of N-linked glycoproteins, alpha-2-HS-glycoprotein and hemopexin: Implications for small molecule compound-mediated neuroprotection

doi: 10.1371/journal.pone.0186227

Figure Lengend Snippet: Human WN1316-activating factors exert WN1316-mediated cytoprotective activity. Differentiated SH-SY5Y cells were treated with 8 μM WN1316 in the presence of purified GST-fusion proteins for 3 h followed by 12 h of chase incubation without the compound, and then exposed to 40 μM menadione for 4 h. Concentrations of the GST-fusion proteins used are as follows; GST; 4.4 μg/ml, GST-RBP4; 2.2 μg/ml, GST-AHSG; 1.9 μg/ml, GST- SERPINA1; 2.4 μg/ml, GST-ITIH4; 2.3 μg/ml, GST-A1BG; 1.5 μg/ml, GST-HPX; 1.7 μg/ml, GST-CFB; 3.3 μg/ml. As control experiments, differentiated SH-SY5Y cells were treated with 8 μM WN1316 (as a positive control) or DMSO (as a vehicle control) in DMEM supplemented with 2% FBS (corresponds to a protein concentration of approximately 0.5 mg/ml). The cell viability was calculated by AlamarBlue, and was expressed as a relative value (relative cytoprotective activity; -fold) of the WN1316-treated samples for vehicle control (DMSO) set as 1. Data are expressed as mean ± SD (n = 4). Statistical significance was evaluated by one-way ANOVA ( p

Article Snippet: Anti-oxidative stress-induced neuronal cell death (AOND) assay SH-SY5Y cells were grown in DMEM supplemented with 10% non-heat inactivated FBS (HyClone, Thermo Fisher Scientific, Waltham, MA), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in 5% CO2 .

Techniques: Activity Assay, Purification, Incubation, Positive Control, Protein Concentration

Journal: Nature Communications

Article Title: Dynamic protein coronas revealed as a modulator of silver nanoparticle sulphidation in vitro

doi: 10.1038/ncomms11770

Figure Lengend Snippet: Protein coronas modulate sulphide formation. Proposed mechanism of protein corona-modulated nano-Ag 2 S formation at Ag NPs, with hard corona proteins trapping Ag + released from the nanoparticle surface and soft corona proteins transporting said ions away from the sulphide-formation centres in the long-lived corona ( a ); TEM images of silver nanocubes after 24 h in RPMI-1640 cell culture medium supplemented with 1% FBS ( b ), followed by 6 days incubation in RPMI-1640 with 0% FBS (inset cartoon showing only hard corona around Ag NPs) ( c ), 1% FBS ( d ) or 10% FBS ( e ; common inset cartoon showing hard and soft coronas, as well as free bulk proteins around Ag NPs); TEM images of silver nanocubes after 7 days incubation in RPMI-1640 with 0.4 mg ml −1 BSA ( f ) or 4 mg ml −1 BSA ( g ) (common inset cartoon showing hard corona and free bulk proteins around Ag NPs); Ultraviolet–visible spectra of cubic ( h ) and quasi-spherical ( i ) Ag NPs after 24 h incubation in RPMI-1640 cell culture medium supplemented with 1, 10 or 50% FBS; TEM images of silver nanocubes after 24 h in RPMI-1640 supplemented with 1% FBS ( j ), 10% FBS ( k ) and 50% FBS ( l ). Scale bars are 100 nm ( b , j – l ) or 50 nm ( c – g ).

Article Snippet: We incubated Ag NPs (cubic or quasi-spherical) in RPMI-1640 with or without added supplements of heat-inactivated FBS (HyClone; 1–50% by volume), for 1 or 7 days.

Techniques: Transmission Electron Microscopy, Cell Culture, Incubation

Journal: Nature Communications

Article Title: Dynamic protein coronas revealed as a modulator of silver nanoparticle sulphidation in vitro

doi: 10.1038/ncomms11770

Figure Lengend Snippet: Silver sulphide forms close to the surface of Ag NPs. TEM image with arrows highlighting nano-Ag 2 S ( a , scale bar 50 nm), X-rays elemental mapping of Ag (red), S (blue, with white rings marking the approximate contour of the Ag NPs) and overlaid Ag and S ( b ), EDS spectrum—with arrows pointing at the peaks corresponding to each element—( c ) and diffraction pattern—arrow pointing at the diffraction line corresponding to monoclinic Ag 2 S—( d ) of silver nanocubes after 7 days incubation in RPMI-1640 supplemented with 1% FBS and formation of Ag 2 S at the surface of the Ag NPs.

Article Snippet: We incubated Ag NPs (cubic or quasi-spherical) in RPMI-1640 with or without added supplements of heat-inactivated FBS (HyClone; 1–50% by volume), for 1 or 7 days.

Techniques: Transmission Electron Microscopy, Incubation

Journal: Nature Communications

Article Title: Dynamic protein coronas revealed as a modulator of silver nanoparticle sulphidation in vitro

doi: 10.1038/ncomms11770

Figure Lengend Snippet: Silver nanoparticle dissolution is involved in nano-Ag 2 S formation. Ultraviolet–visible full spectra of quasi-spherical Ag NPs ( a ) and quadrupole peak detail of nanocubes ( b ) incubated (1 day: blue or 7 days: pink) in RPMI-1640 cell culture medium supplemented with 1% FBS, with or without added extra 10% (by mass) Ag + ions from AgNO 3 ; EDS spectra of the supernatant obtained after centrifugation of Ag NPs incubated for 7 days in RPMI-1640 with 1% FBS, before ( c ) and after ( d ) spiking with 5 nm PVP-coated Ag NPs, with dotted red line highlighting the presence of a silver signal only in the spiked sample.

Article Snippet: We incubated Ag NPs (cubic or quasi-spherical) in RPMI-1640 with or without added supplements of heat-inactivated FBS (HyClone; 1–50% by volume), for 1 or 7 days.

Techniques: Incubation, Cell Culture, Centrifugation

Journal: Nature Communications

Article Title: Dynamic protein coronas revealed as a modulator of silver nanoparticle sulphidation in vitro

doi: 10.1038/ncomms11770

Figure Lengend Snippet: Sulphur sources and the Ag:S ratio influence Ag 2 S formation. TEM images of Ag NPs after 7 days incubation in PBS ( a ), PBS supplemented with 1% FBS ( b ) and PBS supplemented with L -cysteine and L -methionine at the same concentrations of amino acids as those found in RPMI-1640 ( c ) and corresponding EDS spectra ( d ); TEM images and corresponding EDS spectra (insets) of Ag NPs after 7 days incubation in RPMI-1640 supplemented with 1% FBS, with initial silver concentrations of 2 μg ml −1 ( e ), 10 μg ml −1 (f) and 100 μg ml −1 ( g ), with elemental mapping images provided in Supplementary Fig. 26 . Scale bars are 100 nm ( a – c ) or 50 nm ( e – g ).

Article Snippet: We incubated Ag NPs (cubic or quasi-spherical) in RPMI-1640 with or without added supplements of heat-inactivated FBS (HyClone; 1–50% by volume), for 1 or 7 days.

Techniques: Transmission Electron Microscopy, Incubation

Journal: Nature Communications

Article Title: Dynamic protein coronas revealed as a modulator of silver nanoparticle sulphidation in vitro

doi: 10.1038/ncomms11770

Figure Lengend Snippet: Corona-mediated sulphidation of Ag NPs impacts particle toxicity. TEM images of partially sulphidated Ag NPs after pre-incubation in RPMI-1640 with 10% FBS ( a ) and completely sulphidated Ag NPs after pre-incubation in RPMI-1640 with 1% FBS ( b ); scale bars are 50 nm; Viability of J774 murine macrophages (as measured with MTT assays) after 24 h exposure to various concentrations (2, 5, 10, 15, 25, 50 and 100 μg ml −1 ) of Ag + ions (black diamonds), pristine Ag NPs (red triangles), partially sulphidated Ag NPs (blue squares) and completely sulphidated Ag NPs (orange circles); error bars are provided as standard deviation; statistically significant differences (two-tailed t -test, with all data sets showing normal distribution and similar variance values) as compared with the control are marked with ** P

Article Snippet: We incubated Ag NPs (cubic or quasi-spherical) in RPMI-1640 with or without added supplements of heat-inactivated FBS (HyClone; 1–50% by volume), for 1 or 7 days.

Techniques: Transmission Electron Microscopy, Incubation, MTT Assay, Standard Deviation, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Dishevelled-KSRP complex regulates Wnt signaling through post-transcriptional stabilization of β-catenin mRNA

doi: 10.1242/jcs.056176

Figure Lengend Snippet: KSRP overexpression effects β-catenin signaling in colon carcinoma cells. SW480 cells (2.5×10 5 cells/well in a 12-well cell culture dish) were transfected with indicated amounts of either empty vector or FLAG-KSRP plasmid for 24 hours using a Fugene transfection reagent (Roche applied life sciences) to DNA ratio of 5:2. The lysates were then assayed either for cytosolic β-catenin levels ( A ) or Lef/Tcf-sensitive transcription ( B ). Upper panel in B displays mean values ± s.e.m. obtained from three independent experiments; the lower panel displays representative blots. For the β-catenin assay representative blots of two independent experiments that proved highly reproducible are displayed. ## P

Article Snippet: Mouse F9 teratocarcinoma, HEK293 and SW480 cell stocks were obtained from ATCC (Manassas, VA) and were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with either 15% (F9 cells) or 10% (HEK293 cells and SW480) heat-inactivated fetal bovine serum (Hyclone, South Logan, UT) at 37°C in a 5% CO2 incubator.

Techniques: Over Expression, Cell Culture, Transfection, Plasmid Preparation