Structured Review

Lonza fetal bovine serum fbs
Serum deprivation causes selective cytotoxicity, caspase 3/7 activation, and increased lactate formation in G93ASOD1 cells. a The viability of the NSC-34, WT-NSC, and G93A-NSC cell lines was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay after culture with or without 5 % fetal bovine serum <t>(FBS)</t> for 22 h. Percentages of the MTT conversion after culture with serum (100 %; n = 12) are shown. b Activation of caspase 3/7 of the NSC-34, WT-NSC, and G93A-NSC cell lines cultured without 5 % serum for 17 h. Values are percentages of the NSC-34 activity (100 %; n = 15). c Levels of lactate in the medium (μmol/mg of protein; n = 8) of the NSC-34, WT-NSC, and G93A-NSC cell lines cultured with or without 5 % serum for 22 h. All values are mean ± s.e.m. For each parameter, statistical significance of differences was assessed by one-way ANOVA and Tukey’s post hoc test comparing the levels of the different cell lines with or without 5 % serum (respectively ### p
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1) Product Images from "Metabolomic Analysis Reveals Increased Aerobic Glycolysis and Amino Acid Deficit in a Cellular Model of Amyotrophic Lateral Sclerosis"

Article Title: Metabolomic Analysis Reveals Increased Aerobic Glycolysis and Amino Acid Deficit in a Cellular Model of Amyotrophic Lateral Sclerosis

Journal: Molecular Neurobiology

doi: 10.1007/s12035-015-9165-7

Serum deprivation causes selective cytotoxicity, caspase 3/7 activation, and increased lactate formation in G93ASOD1 cells. a The viability of the NSC-34, WT-NSC, and G93A-NSC cell lines was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay after culture with or without 5 % fetal bovine serum (FBS) for 22 h. Percentages of the MTT conversion after culture with serum (100 %; n = 12) are shown. b Activation of caspase 3/7 of the NSC-34, WT-NSC, and G93A-NSC cell lines cultured without 5 % serum for 17 h. Values are percentages of the NSC-34 activity (100 %; n = 15). c Levels of lactate in the medium (μmol/mg of protein; n = 8) of the NSC-34, WT-NSC, and G93A-NSC cell lines cultured with or without 5 % serum for 22 h. All values are mean ± s.e.m. For each parameter, statistical significance of differences was assessed by one-way ANOVA and Tukey’s post hoc test comparing the levels of the different cell lines with or without 5 % serum (respectively ### p
Figure Legend Snippet: Serum deprivation causes selective cytotoxicity, caspase 3/7 activation, and increased lactate formation in G93ASOD1 cells. a The viability of the NSC-34, WT-NSC, and G93A-NSC cell lines was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay after culture with or without 5 % fetal bovine serum (FBS) for 22 h. Percentages of the MTT conversion after culture with serum (100 %; n = 12) are shown. b Activation of caspase 3/7 of the NSC-34, WT-NSC, and G93A-NSC cell lines cultured without 5 % serum for 17 h. Values are percentages of the NSC-34 activity (100 %; n = 15). c Levels of lactate in the medium (μmol/mg of protein; n = 8) of the NSC-34, WT-NSC, and G93A-NSC cell lines cultured with or without 5 % serum for 22 h. All values are mean ± s.e.m. For each parameter, statistical significance of differences was assessed by one-way ANOVA and Tukey’s post hoc test comparing the levels of the different cell lines with or without 5 % serum (respectively ### p

Techniques Used: Activation Assay, MTT Assay, Cell Culture, Activity Assay

2) Product Images from "17?-Estradiol Enhances Signalling Mediated by VEGF-A-Delta-Like Ligand 4-Notch1 Axis in Human Endothelial Cells"

Article Title: 17?-Estradiol Enhances Signalling Mediated by VEGF-A-Delta-Like Ligand 4-Notch1 Axis in Human Endothelial Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0071440

Effect of 17β-estradiol treatment on Notch ligands in HUVECs under different experimental conditions. (A) HUVECs were treated with 1 nM E2 or DMSO (V) for 24 hours under M4 (2% FBS overnight followed by 20% csFBS) or M5 (2% csFBS-EGM-2) experimental conditions. Cell lysates were electrophoresed and immunoblotted with Jagged1, Dll4 and Dll1 antibodies. β-actin antibody was used to ensure equal loading. Densitometric analysis of Western blot assay is shown in Figure S6E . (B) HUVECs were exposed to different VEGF-A concentrations (20 ng/ml and 50 ng/ml) for 24 hours under M5 experimental conditions (2% csFBS-EGM-2). Cell lysates were electrophoresed and immunoblotted with Dll4 antibody. β-actin antibody was used to ensure equal loading. Densitometric analysis of Western blot assay is shown in Figure S6F . (C) HUVECs were treated with VEGF-A (20 ng/ml and 50 ng/ml) for 24 hours under M5 experimental conditions. Total RNA was extracted and qRT-PCR analysis of Dll4 gene expression was performed. Relative changes in mRNA expression levels were calculated according to the 2 −ΔΔCt method using RPL13A as reference gene. Results are expressed as mean ± SEM of three independent experiments, each performed in triplicate. *** P
Figure Legend Snippet: Effect of 17β-estradiol treatment on Notch ligands in HUVECs under different experimental conditions. (A) HUVECs were treated with 1 nM E2 or DMSO (V) for 24 hours under M4 (2% FBS overnight followed by 20% csFBS) or M5 (2% csFBS-EGM-2) experimental conditions. Cell lysates were electrophoresed and immunoblotted with Jagged1, Dll4 and Dll1 antibodies. β-actin antibody was used to ensure equal loading. Densitometric analysis of Western blot assay is shown in Figure S6E . (B) HUVECs were exposed to different VEGF-A concentrations (20 ng/ml and 50 ng/ml) for 24 hours under M5 experimental conditions (2% csFBS-EGM-2). Cell lysates were electrophoresed and immunoblotted with Dll4 antibody. β-actin antibody was used to ensure equal loading. Densitometric analysis of Western blot assay is shown in Figure S6F . (C) HUVECs were treated with VEGF-A (20 ng/ml and 50 ng/ml) for 24 hours under M5 experimental conditions. Total RNA was extracted and qRT-PCR analysis of Dll4 gene expression was performed. Relative changes in mRNA expression levels were calculated according to the 2 −ΔΔCt method using RPL13A as reference gene. Results are expressed as mean ± SEM of three independent experiments, each performed in triplicate. *** P

Techniques Used: Western Blot, Quantitative RT-PCR, Expressing

3) Product Images from "Comparison among Activities and Isoflavonoids from Pueraria thunbergiana Aerial Parts and Root"

Article Title: Comparison among Activities and Isoflavonoids from Pueraria thunbergiana Aerial Parts and Root

Journal: Molecules

doi: 10.3390/molecules24050912

Effects by extraction parts of kudzu on the viability and NO levels of RAW 264.7 macrophages. A density of 1 × 10 4 cells/well of macrophages were seeded in a 96-well plate and incubated with various concentrations of each extract for 24 h and cell viability was determined by MTT assay. A density of 1 × 10 4 cells/well of macrophages were seeded in a 96-well plate and the various concentrations of sample and LPS (1 μg/mL) were treated with DMEM (without penicillin and fetal bovine serum (FBS)) to 0.2 mL/well and incubated for 24 h. Untreated samples without LPS treatment were negative controls. Each value is expressed as mean ± SD of three independent experiments. *; p
Figure Legend Snippet: Effects by extraction parts of kudzu on the viability and NO levels of RAW 264.7 macrophages. A density of 1 × 10 4 cells/well of macrophages were seeded in a 96-well plate and incubated with various concentrations of each extract for 24 h and cell viability was determined by MTT assay. A density of 1 × 10 4 cells/well of macrophages were seeded in a 96-well plate and the various concentrations of sample and LPS (1 μg/mL) were treated with DMEM (without penicillin and fetal bovine serum (FBS)) to 0.2 mL/well and incubated for 24 h. Untreated samples without LPS treatment were negative controls. Each value is expressed as mean ± SD of three independent experiments. *; p

Techniques Used: Incubation, MTT Assay

4) Product Images from "Chenodeoxycholic Acid Reduces Hypoxia Inducible Factor-1α Protein and Its Target Genes"

Article Title: Chenodeoxycholic Acid Reduces Hypoxia Inducible Factor-1α Protein and Its Target Genes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0130911

Effect of CDCA on hypoxic target genes. (A) Experimental scheme. HepG2 cells were serum starved with medium containing 0.5% FBS for 20 hours prior to CDCA (100 μM or indicated dose) treatment. 6 hours after CDCA treatment, the cells were exposed to 20%, 5% or 0.1% O 2 for the indicated hours. (B) Quantitative RT-PCR analyses of SHP mRNA. The expression level was normalized with the expression level of 18s rRNA. (C) Western analyses for SHP and β-actin. β-actin protein was detected as a loading control. Data shown are representative of three experiments (C) Quantitative RT-PCR analyses of carbonic anhydrase 9 (CA9), phosphoglycerate kinase1 (PGK1), endoplasmic reticulum oxidoreductin 1-like (EROL1), lysyl oxidase (LOX), prolyl 4-hydroxylase, alpha peptide 1 (P4HA1). a, p ≤ 0.1; b, p ≤ 0.05; c, p ≤ 0.01; d, p ≤ 0.001; e, p = 0.197; f, p = 0.724.
Figure Legend Snippet: Effect of CDCA on hypoxic target genes. (A) Experimental scheme. HepG2 cells were serum starved with medium containing 0.5% FBS for 20 hours prior to CDCA (100 μM or indicated dose) treatment. 6 hours after CDCA treatment, the cells were exposed to 20%, 5% or 0.1% O 2 for the indicated hours. (B) Quantitative RT-PCR analyses of SHP mRNA. The expression level was normalized with the expression level of 18s rRNA. (C) Western analyses for SHP and β-actin. β-actin protein was detected as a loading control. Data shown are representative of three experiments (C) Quantitative RT-PCR analyses of carbonic anhydrase 9 (CA9), phosphoglycerate kinase1 (PGK1), endoplasmic reticulum oxidoreductin 1-like (EROL1), lysyl oxidase (LOX), prolyl 4-hydroxylase, alpha peptide 1 (P4HA1). a, p ≤ 0.1; b, p ≤ 0.05; c, p ≤ 0.01; d, p ≤ 0.001; e, p = 0.197; f, p = 0.724.

Techniques Used: Quantitative RT-PCR, Expressing, Western Blot

Effects of SHP or GW4064 on HIF-1α expression. (A and B) HepG2 cells were transfected with an empty vector or pcDNA3/HA-SHP. 18 hours after transfection, the cells were serum starved with medium containing 0.5% FBS for 20 hours. The cells were treated DMSO or 100 μM of CDCA for 6 hours in the absence or presence of MG132 and then exposed to 20%, 5% or 0.1% O 2 for 4 hours. 30 μg of total cell extracts are loaded for western analyses. (C to E) After starvation for 20hr, HepG2 cells were pretreated with GW4064 (GW) (5 μM or indicated dose) for 6 hours then exposed to 20%, 5% or 0.1% O 2 for the indicated hours. (C and E) qRT-PCR analyses of SHP or HIF-1α respectively. The expression level was normalized with the expression level of 18s rRNA. a, p ≤ 0.1; b, p ≤ 0.05; c, p ≤ 0.01; d, p ≤ 0.001. (D) Western analyses of HIF-1α, SHP and β-actin. (F) Western analyses of HIF-1α and β-actin in HepG2 cells which were treated with indicated doses of GW4064 and MG132 as described above. Ubiquitinated and original HIF-1α proteins are indicated. β-actin protein were examined in order to verify equal loading.
Figure Legend Snippet: Effects of SHP or GW4064 on HIF-1α expression. (A and B) HepG2 cells were transfected with an empty vector or pcDNA3/HA-SHP. 18 hours after transfection, the cells were serum starved with medium containing 0.5% FBS for 20 hours. The cells were treated DMSO or 100 μM of CDCA for 6 hours in the absence or presence of MG132 and then exposed to 20%, 5% or 0.1% O 2 for 4 hours. 30 μg of total cell extracts are loaded for western analyses. (C to E) After starvation for 20hr, HepG2 cells were pretreated with GW4064 (GW) (5 μM or indicated dose) for 6 hours then exposed to 20%, 5% or 0.1% O 2 for the indicated hours. (C and E) qRT-PCR analyses of SHP or HIF-1α respectively. The expression level was normalized with the expression level of 18s rRNA. a, p ≤ 0.1; b, p ≤ 0.05; c, p ≤ 0.01; d, p ≤ 0.001. (D) Western analyses of HIF-1α, SHP and β-actin. (F) Western analyses of HIF-1α and β-actin in HepG2 cells which were treated with indicated doses of GW4064 and MG132 as described above. Ubiquitinated and original HIF-1α proteins are indicated. β-actin protein were examined in order to verify equal loading.

Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR

Effect of CDCA on HIF-1α expression. After starvation for 20hr, HepG2 cells were pretreated with CDCA (100 μM or indicated dose) for 6 hours then exposed to 20%, 5% or 0.1% O 2 for the indicated hours. (A) Western analyses for HIF-1α, 14-3-3γ and β-actin proteins. 14-3-3γ and β-actin proteins were detected as loading controls. (B) Quantitative RT-PCR of HIF-1α mRNA. (C and D) Western analyses of HIF-1α protein. HepG2 cells which were serum starved with medium containing 0.5% FBS for 20 hours prior to stimulation with MG132 (10 μM) and/or CDCA. 6 hours after treatment, the cells were exposed to 20% or 5% O 2 for 4 hours. Ubiquitinated and original HIF-1α proteins are indicated. HDAC1 protein or β-actin protein were examined in order to verify equal loading. (D) 20 μg of MG132 untreated total cell extracts and 5 μg of MG132 treated total cell extracts are loaded, respectively.
Figure Legend Snippet: Effect of CDCA on HIF-1α expression. After starvation for 20hr, HepG2 cells were pretreated with CDCA (100 μM or indicated dose) for 6 hours then exposed to 20%, 5% or 0.1% O 2 for the indicated hours. (A) Western analyses for HIF-1α, 14-3-3γ and β-actin proteins. 14-3-3γ and β-actin proteins were detected as loading controls. (B) Quantitative RT-PCR of HIF-1α mRNA. (C and D) Western analyses of HIF-1α protein. HepG2 cells which were serum starved with medium containing 0.5% FBS for 20 hours prior to stimulation with MG132 (10 μM) and/or CDCA. 6 hours after treatment, the cells were exposed to 20% or 5% O 2 for 4 hours. Ubiquitinated and original HIF-1α proteins are indicated. HDAC1 protein or β-actin protein were examined in order to verify equal loading. (D) 20 μg of MG132 untreated total cell extracts and 5 μg of MG132 treated total cell extracts are loaded, respectively.

Techniques Used: Expressing, Western Blot, Quantitative RT-PCR

5) Product Images from "Grb2 depletion under non-stimulated conditions inhibits PTEN, promotes Akt-induced tumor formation and contributes to poor prognosis in ovarian cancer"

Article Title: Grb2 depletion under non-stimulated conditions inhibits PTEN, promotes Akt-induced tumor formation and contributes to poor prognosis in ovarian cancer

Journal: Oncogene

doi: 10.1038/onc.2015.279

Akt pathway-dependent colony formation is enhanced in Grb2 knockdown cells (a) HEK293T cells with and without FGFR2 were transfected with scrambled shRNA, Grb2 shRNA and/or Plcγ1 shRNA to generate stable knockdowns: PCi, PG2i and PPγi for parental cells, and Ci, G2i, Pγi and Grb2/Pγi (double knockdown, DKD) for cells transfected with GFP-FGFR2. 0.6 × 10 4 cells were cast as a single cell suspension in serum-deprived DMEM/agar in the upper layer with DMEM/agar with 1% FBS in the lower layer. Cells were left to grow for 8 days. DMEM with 1% FBS was added to the wells once every seven days. Pictures of the entire wells were taken. Pictures of colonies made from Ci or G2i cells transfected with GFP-FGFR2 were taken at 20X magnification to show the difference in colony size formed. (b) Colonies formed in each case in (a) were counted and averaged in 4 microscopic fields (independent experiments n=3). Error bars on the graph represent the standard deviation of the calculated mean. Student's t-test indicates p≤ 0.01 which denotes a significant increase in the number of colonies formed upon Grb2 depletion in FGR2 expressing cells. , f-I for efficiency of inhibition. (d) Colonies formed in each case in (c) were counted and averaged in 4 microscopic fields (independent experiments n=3). Error bars on the graph represent the standard deviation of the calculated mean. Student's t-test indicates *p≤ 0.05 and **p≤ 0.01. . (f) Results of Akt in vitro .
Figure Legend Snippet: Akt pathway-dependent colony formation is enhanced in Grb2 knockdown cells (a) HEK293T cells with and without FGFR2 were transfected with scrambled shRNA, Grb2 shRNA and/or Plcγ1 shRNA to generate stable knockdowns: PCi, PG2i and PPγi for parental cells, and Ci, G2i, Pγi and Grb2/Pγi (double knockdown, DKD) for cells transfected with GFP-FGFR2. 0.6 × 10 4 cells were cast as a single cell suspension in serum-deprived DMEM/agar in the upper layer with DMEM/agar with 1% FBS in the lower layer. Cells were left to grow for 8 days. DMEM with 1% FBS was added to the wells once every seven days. Pictures of the entire wells were taken. Pictures of colonies made from Ci or G2i cells transfected with GFP-FGFR2 were taken at 20X magnification to show the difference in colony size formed. (b) Colonies formed in each case in (a) were counted and averaged in 4 microscopic fields (independent experiments n=3). Error bars on the graph represent the standard deviation of the calculated mean. Student's t-test indicates p≤ 0.01 which denotes a significant increase in the number of colonies formed upon Grb2 depletion in FGR2 expressing cells. , f-I for efficiency of inhibition. (d) Colonies formed in each case in (c) were counted and averaged in 4 microscopic fields (independent experiments n=3). Error bars on the graph represent the standard deviation of the calculated mean. Student's t-test indicates *p≤ 0.05 and **p≤ 0.01. . (f) Results of Akt in vitro .

Techniques Used: Transfection, shRNA, Standard Deviation, Expressing, Inhibition, In Vitro

6) Product Images from "Carnosic Acid Inhibits the Epithelial-Mesenchymal Transition in B16F10 Melanoma Cells: A Possible Mechanism for the Inhibition of Cell Migration"

Article Title: Carnosic Acid Inhibits the Epithelial-Mesenchymal Transition in B16F10 Melanoma Cells: A Possible Mechanism for the Inhibition of Cell Migration

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms150712698

Carnosic acid inhibits migration of B16F10 melanoma cells. ( A ) Structure of carnosic acid; ( B – D ) Transwell migration assays were conducted with Lewis lung cancer (LLC) cells (2.5 × 10 4 cells/well) ( B ); CT26 cells (2.5 × 10 4 cells/well) ( C ); and B16F10 cells (5 × 10 4 cells/well) ( D ) Migrating cells were quantified, and each bar represents the mean ± SEM from three independent experiments; ( E ) B16F10 cells were plated in 100 mm dishes at 1 × 10 6 cells/dish in MEM supplemented with 10% FBS and 292 mg/L l -glutamine. After 12 h of incubation, the monolayers were serum-starved in MEM then treated for 12 h with carnosic acid. Conditioned media were collected and concentrated for Western blotting. The volumes of media loaded onto the gel were adjusted for equivalent proteins. Photographs of chemiluminescent detection of the immunoblots, which are representative of three independent experiments, are shown. Relative abundance of each band was estimated by densitometric scanning of the exposed films. The adjusted mean ± SEM ( n = 3) of each band is shown above each blot. * Significantly different from the control (0 μmol/L carnosic acid), p
Figure Legend Snippet: Carnosic acid inhibits migration of B16F10 melanoma cells. ( A ) Structure of carnosic acid; ( B – D ) Transwell migration assays were conducted with Lewis lung cancer (LLC) cells (2.5 × 10 4 cells/well) ( B ); CT26 cells (2.5 × 10 4 cells/well) ( C ); and B16F10 cells (5 × 10 4 cells/well) ( D ) Migrating cells were quantified, and each bar represents the mean ± SEM from three independent experiments; ( E ) B16F10 cells were plated in 100 mm dishes at 1 × 10 6 cells/dish in MEM supplemented with 10% FBS and 292 mg/L l -glutamine. After 12 h of incubation, the monolayers were serum-starved in MEM then treated for 12 h with carnosic acid. Conditioned media were collected and concentrated for Western blotting. The volumes of media loaded onto the gel were adjusted for equivalent proteins. Photographs of chemiluminescent detection of the immunoblots, which are representative of three independent experiments, are shown. Relative abundance of each band was estimated by densitometric scanning of the exposed films. The adjusted mean ± SEM ( n = 3) of each band is shown above each blot. * Significantly different from the control (0 μmol/L carnosic acid), p

Techniques Used: Migration, Incubation, Western Blot

7) Product Images from "Stem cell exosomes inhibit angiogenesis and tumor growth of oral squamous cell carcinoma"

Article Title: Stem cell exosomes inhibit angiogenesis and tumor growth of oral squamous cell carcinoma

Journal: Scientific Reports

doi: 10.1038/s41598-018-36855-6

Carrier-based cell culture increases exosome yield. ( A ) Schematic overview of exosome production process. MenSC were expanded on 2D cell culture dishes and then seeded on BioNOC II. After 72 h in DMEM with 10% FBS, the medium was changed to serum-free DMEM for 72 h for exosome production. For purification, the supernatant was collected and processed in serial centrifugations. ( B ) Western Blot with 15 µg of cell lysate (Cells) and exosomes (Exo). To confirm the purity of the exosomes, positive exosomal markers CD63, Syntenin and CD9 and negative exosomal markers Vinculin (Vinc.) and Calreticulin (Calr.) and β-Actin (actin) were analyzed. ( C ) Scanning electron micrograph of purified exosomes, magnification 60,000x. ( D ) Size distribution of exosomes determined by nanosight showing that the highest abundance of particles was below 200 nm. ( E ) Hoechst-stained MenSC on BioNOC II carrier, showing a typical confluence for exosome production. ( F ) Yield of purified exosomes in PBS as Particles (part)/ml of initial cell culture supernatant (SN) are shown for 2D (N = 32) and 3D culture (N = 10). Analysis: unpaired t-test. Bar graphs show average values, error bars: SEM.
Figure Legend Snippet: Carrier-based cell culture increases exosome yield. ( A ) Schematic overview of exosome production process. MenSC were expanded on 2D cell culture dishes and then seeded on BioNOC II. After 72 h in DMEM with 10% FBS, the medium was changed to serum-free DMEM for 72 h for exosome production. For purification, the supernatant was collected and processed in serial centrifugations. ( B ) Western Blot with 15 µg of cell lysate (Cells) and exosomes (Exo). To confirm the purity of the exosomes, positive exosomal markers CD63, Syntenin and CD9 and negative exosomal markers Vinculin (Vinc.) and Calreticulin (Calr.) and β-Actin (actin) were analyzed. ( C ) Scanning electron micrograph of purified exosomes, magnification 60,000x. ( D ) Size distribution of exosomes determined by nanosight showing that the highest abundance of particles was below 200 nm. ( E ) Hoechst-stained MenSC on BioNOC II carrier, showing a typical confluence for exosome production. ( F ) Yield of purified exosomes in PBS as Particles (part)/ml of initial cell culture supernatant (SN) are shown for 2D (N = 32) and 3D culture (N = 10). Analysis: unpaired t-test. Bar graphs show average values, error bars: SEM.

Techniques Used: Cell Culture, Purification, Western Blot, Staining

8) Product Images from "Roles of Tetrahydrobiopterin in Promoting Tumor Angiogenesis"

Article Title: Roles of Tetrahydrobiopterin in Promoting Tumor Angiogenesis

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2010.100025

Phosphorylation of eNOS and Akt by BH4 synthesis depends on PI3K signaling in COS-7 cells and HUVEC. After overnight culture without FBS, human eNOS-transfected COS-7 cell monolayers were incubated for 30 minutes with Sep at various doses (0, 2, 5, and
Figure Legend Snippet: Phosphorylation of eNOS and Akt by BH4 synthesis depends on PI3K signaling in COS-7 cells and HUVEC. After overnight culture without FBS, human eNOS-transfected COS-7 cell monolayers were incubated for 30 minutes with Sep at various doses (0, 2, 5, and

Techniques Used: Transfection, Incubation

9) Product Images from "A Novel Peptide to Treat Oral Mucositis Blocks Endothelial and Epithelial Cell Apoptosis"

Article Title: A Novel Peptide to Treat Oral Mucositis Blocks Endothelial and Epithelial Cell Apoptosis

Journal: International Journal of Radiation Oncology, Biology, Physics

doi: 10.1016/j.ijrobp.2012.01.006

Antiapoptotic effects of antrum mucosal protein peptide (AMP-p) in human dermal microvascular endothelial cells (HDMECs) exposed to tumor necrosis factor (TNF) α. HDMECs in Dulbecco's modified Eagle's medium (DMEM) plus 5% fetal bovine serum (FBS)
Figure Legend Snippet: Antiapoptotic effects of antrum mucosal protein peptide (AMP-p) in human dermal microvascular endothelial cells (HDMECs) exposed to tumor necrosis factor (TNF) α. HDMECs in Dulbecco's modified Eagle's medium (DMEM) plus 5% fetal bovine serum (FBS)

Techniques Used: Modification

10) Product Images from "MEF2 Is a Converging Hub for Histone Deacetylase 4 and Phosphatidylinositol 3-Kinase/Akt-Induced Transformation"

Article Title: MEF2 Is a Converging Hub for Histone Deacetylase 4 and Phosphatidylinositol 3-Kinase/Akt-Induced Transformation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01050-13

Transforming ability of HDAC4/TM. (A) NIH 3T3 cells expressing the indicated transgenes were grown in DMEM supplemented with 10% FBS. (B) Growth in soft agar of NIH 3T3 expressing the indicated transgenes, foci were stained with MTT. (C) Quantitative
Figure Legend Snippet: Transforming ability of HDAC4/TM. (A) NIH 3T3 cells expressing the indicated transgenes were grown in DMEM supplemented with 10% FBS. (B) Growth in soft agar of NIH 3T3 expressing the indicated transgenes, foci were stained with MTT. (C) Quantitative

Techniques Used: Expressing, Staining, MTT Assay

11) Product Images from "Microvascular Mural Cell Functionality of Human Embryonic Stem Cell-Derived Mesenchymal Cells"

Article Title: Microvascular Mural Cell Functionality of Human Embryonic Stem Cell-Derived Mesenchymal Cells

Journal: Tissue Engineering. Part A

doi: 10.1089/ten.tea.2010.0397

TGFβ1 induces αSMA expression under serum-free conditions. (A) hES-MC were grown to confluence in chamber slides and cultured for 2 days in SFM, after which cells were maintained in SFM alone (top left), addition of 10% FBS (bottom left),
Figure Legend Snippet: TGFβ1 induces αSMA expression under serum-free conditions. (A) hES-MC were grown to confluence in chamber slides and cultured for 2 days in SFM, after which cells were maintained in SFM alone (top left), addition of 10% FBS (bottom left),

Techniques Used: Expressing, Cell Culture

12) Product Images from "Procyanidin B2 3,3″-di-O-gallate inhibits endothelial cells growth and motility by targeting VEGFR2 and integrin signaling pathways"

Article Title: Procyanidin B2 3,3″-di-O-gallate inhibits endothelial cells growth and motility by targeting VEGFR2 and integrin signaling pathways

Journal: Current cancer drug targets

doi:

Effect of B2G2 on Prostate cancer cells-induced capillary tube formation by HUVECs. (A) LNCaP and PC3 cells were treated with B2G2 (30 μM) and grown under normoxic (~21% O 2 ) and hypoxic (1% O 2 ) conditions and conditioned media was collected as described in ‘Materials and Methods’. HUVECs were seeded on matrigel along with 0.5% FBS containing media or conditioned media and EBM-2 (75:25 ratio) and capillary tube formation was analyzed after 6 h. Representative images depicting formation of capillary tubes are shown at 100x magnification. In this experiment, HUVECs incubated with 0.5% FBS containing LNCaP or PC-3 media alone served as a negative control. (B) Tube length was measured as described in ‘Materials and Methods’. Tube length data is presented as mean ± standard error of three samples for each treatment. *p
Figure Legend Snippet: Effect of B2G2 on Prostate cancer cells-induced capillary tube formation by HUVECs. (A) LNCaP and PC3 cells were treated with B2G2 (30 μM) and grown under normoxic (~21% O 2 ) and hypoxic (1% O 2 ) conditions and conditioned media was collected as described in ‘Materials and Methods’. HUVECs were seeded on matrigel along with 0.5% FBS containing media or conditioned media and EBM-2 (75:25 ratio) and capillary tube formation was analyzed after 6 h. Representative images depicting formation of capillary tubes are shown at 100x magnification. In this experiment, HUVECs incubated with 0.5% FBS containing LNCaP or PC-3 media alone served as a negative control. (B) Tube length was measured as described in ‘Materials and Methods’. Tube length data is presented as mean ± standard error of three samples for each treatment. *p

Techniques Used: Incubation, Negative Control

13) Product Images from "Pak2 regulates hematopoietic progenitor cell proliferation, survival and differentiation"

Article Title: Pak2 regulates hematopoietic progenitor cell proliferation, survival and differentiation

Journal: Stem cells (Dayton, Ohio)

doi: 10.1002/stem.1951

Pak2 regulates hematopoietic progenitor cell proliferation, survival and apoptosis Pak2 flox/flox c-kit + BM cells were untransduced or transduced with lentiviral vectors encoding either eGFP ( LVeGFP ) or Cre-eGFP ( LVCre-eGFP ). GFP + cells were sorted and used. (A) Cells were cultured in IMDM+10% FBS+100ng/ml of mSCF for 24 hours before cell cycle analysis. Panels (i) and (ii) demonstrate the pooled G0/G1 phase and S phase data from at least 3 mice, respectively. Student t test, p
Figure Legend Snippet: Pak2 regulates hematopoietic progenitor cell proliferation, survival and apoptosis Pak2 flox/flox c-kit + BM cells were untransduced or transduced with lentiviral vectors encoding either eGFP ( LVeGFP ) or Cre-eGFP ( LVCre-eGFP ). GFP + cells were sorted and used. (A) Cells were cultured in IMDM+10% FBS+100ng/ml of mSCF for 24 hours before cell cycle analysis. Panels (i) and (ii) demonstrate the pooled G0/G1 phase and S phase data from at least 3 mice, respectively. Student t test, p

Techniques Used: Transduction, Cell Culture, Cell Cycle Assay, Mouse Assay

14) Product Images from "Unaltered Angiogenesis-Regulating Activities of Platelets in Mild Type 2 Diabetes Mellitus despite a Marked Platelet Hyperreactivity"

Article Title: Unaltered Angiogenesis-Regulating Activities of Platelets in Mild Type 2 Diabetes Mellitus despite a Marked Platelet Hyperreactivity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0162405

Platelet releasates enhance tube formation of ECFCs. ECFCs (1×10 4 ) suspended in EBM-2 basal medium with 0.5% FBS were seeded onto Matrigel-coated wells of a 96-well plate, and incubated at 37°C for 6 h in presence of vehicle (panel A), 8 μM PAR1-AP (B), 10 μM PAR4-AP (C), 10% PAR1-PR (D and F), or10% PAR4-PR (E and G) of non-diabetic (D and E) and T2DM subjects (F and G). ECFC tube formation was observed and fotographed using an Olympus CKX41 inverted light microscope equipped with a Nikon D5100 camera. The tube formation images were subjected to WimTube tube formation image analysis. Total branch points (H) and total tube length (I) of ECFC tube formation are plotted as mean±SEM; n = 5. *P
Figure Legend Snippet: Platelet releasates enhance tube formation of ECFCs. ECFCs (1×10 4 ) suspended in EBM-2 basal medium with 0.5% FBS were seeded onto Matrigel-coated wells of a 96-well plate, and incubated at 37°C for 6 h in presence of vehicle (panel A), 8 μM PAR1-AP (B), 10 μM PAR4-AP (C), 10% PAR1-PR (D and F), or10% PAR4-PR (E and G) of non-diabetic (D and E) and T2DM subjects (F and G). ECFC tube formation was observed and fotographed using an Olympus CKX41 inverted light microscope equipped with a Nikon D5100 camera. The tube formation images were subjected to WimTube tube formation image analysis. Total branch points (H) and total tube length (I) of ECFC tube formation are plotted as mean±SEM; n = 5. *P

Techniques Used: Incubation, Light Microscopy

Platelets per se enhance tube formation of ECFCs. ECFCs (1×10 4 ) suspended in EBM-2 basal medium with 0.5% FBS were seeded onto Matrigel-coated wells and incubated in presence of vehicle or platelets (ECFC:platelet ratio at 1:200) at 37°C for 6 h. ECFC tube formation was observed and fotographed using an Olympus CKX41 inverted light microscope equipped with a Nikon D5100 camera. The images presented are ECFC tube formation without platelets (panel A), with control platelets (B), and with T2DM platelets (C). Parameters of tube formation were analysed using WimTube tube formation image analysis. Total branch points (D), total tube length (E), total loops (F), and covered area (G) of ECFC tube formation are plotted as mean±SEM; n = 5. **P
Figure Legend Snippet: Platelets per se enhance tube formation of ECFCs. ECFCs (1×10 4 ) suspended in EBM-2 basal medium with 0.5% FBS were seeded onto Matrigel-coated wells and incubated in presence of vehicle or platelets (ECFC:platelet ratio at 1:200) at 37°C for 6 h. ECFC tube formation was observed and fotographed using an Olympus CKX41 inverted light microscope equipped with a Nikon D5100 camera. The images presented are ECFC tube formation without platelets (panel A), with control platelets (B), and with T2DM platelets (C). Parameters of tube formation were analysed using WimTube tube formation image analysis. Total branch points (D), total tube length (E), total loops (F), and covered area (G) of ECFC tube formation are plotted as mean±SEM; n = 5. **P

Techniques Used: Incubation, Light Microscopy

15) Product Images from "Stem cell exosomes inhibit angiogenesis and tumor growth of oral squamous cell carcinoma"

Article Title: Stem cell exosomes inhibit angiogenesis and tumor growth of oral squamous cell carcinoma

Journal: Scientific Reports

doi: 10.1038/s41598-018-36855-6

Carrier-based cell culture increases exosome yield. ( A ) Schematic overview of exosome production process. MenSC were expanded on 2D cell culture dishes and then seeded on BioNOC II. After 72 h in DMEM with 10% FBS, the medium was changed to serum-free DMEM for 72 h for exosome production. For purification, the supernatant was collected and processed in serial centrifugations. ( B ) Western Blot with 15 µg of cell lysate (Cells) and exosomes (Exo). To confirm the purity of the exosomes, positive exosomal markers CD63, Syntenin and CD9 and negative exosomal markers Vinculin (Vinc.) and Calreticulin (Calr.) and β-Actin (actin) were analyzed. ( C ) Scanning electron micrograph of purified exosomes, magnification 60,000x. ( D ) Size distribution of exosomes determined by nanosight showing that the highest abundance of particles was below 200 nm. ( E ) Hoechst-stained MenSC on BioNOC II carrier, showing a typical confluence for exosome production. ( F ) Yield of purified exosomes in PBS as Particles (part)/ml of initial cell culture supernatant (SN) are shown for 2D (N = 32) and 3D culture (N = 10). Analysis: unpaired t-test. Bar graphs show average values, error bars: SEM.
Figure Legend Snippet: Carrier-based cell culture increases exosome yield. ( A ) Schematic overview of exosome production process. MenSC were expanded on 2D cell culture dishes and then seeded on BioNOC II. After 72 h in DMEM with 10% FBS, the medium was changed to serum-free DMEM for 72 h for exosome production. For purification, the supernatant was collected and processed in serial centrifugations. ( B ) Western Blot with 15 µg of cell lysate (Cells) and exosomes (Exo). To confirm the purity of the exosomes, positive exosomal markers CD63, Syntenin and CD9 and negative exosomal markers Vinculin (Vinc.) and Calreticulin (Calr.) and β-Actin (actin) were analyzed. ( C ) Scanning electron micrograph of purified exosomes, magnification 60,000x. ( D ) Size distribution of exosomes determined by nanosight showing that the highest abundance of particles was below 200 nm. ( E ) Hoechst-stained MenSC on BioNOC II carrier, showing a typical confluence for exosome production. ( F ) Yield of purified exosomes in PBS as Particles (part)/ml of initial cell culture supernatant (SN) are shown for 2D (N = 32) and 3D culture (N = 10). Analysis: unpaired t-test. Bar graphs show average values, error bars: SEM.

Techniques Used: Cell Culture, Purification, Western Blot, Staining

16) Product Images from "Bone morphogenetic protein-7 regulates Snail signaling in carbon tetrachloride-induced fibrosis in the rat liver"

Article Title: Bone morphogenetic protein-7 regulates Snail signaling in carbon tetrachloride-induced fibrosis in the rat liver

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2012.720

Ratio of α-SMA (a) and E-cadherin/GAPDH (b) in each group. (A) control group; (B) liver fibrosis model group; (C) BMP-7-treated group. −, Primary rat hepatocytes (1×10 6 /dish) were cultured for 48 h in F12 medium containing 10% fetal bovine serum and 2  μ g/ml insulin until they had adhered. +, Primary rat hepatocytes (1×10 6 /dish) were cultured for 48 h in F12 medium containing 10% fetal bovine serum and 2  μ g/ml insulin until they had adhered. To induce EMT, the media was replaced with F12 media supplemented with 0.5% fetal bovine serum and 200 mg/ μ l insulin containing Snail for 96 h.  * P
Figure Legend Snippet: Ratio of α-SMA (a) and E-cadherin/GAPDH (b) in each group. (A) control group; (B) liver fibrosis model group; (C) BMP-7-treated group. −, Primary rat hepatocytes (1×10 6 /dish) were cultured for 48 h in F12 medium containing 10% fetal bovine serum and 2 μ g/ml insulin until they had adhered. +, Primary rat hepatocytes (1×10 6 /dish) were cultured for 48 h in F12 medium containing 10% fetal bovine serum and 2 μ g/ml insulin until they had adhered. To induce EMT, the media was replaced with F12 media supplemented with 0.5% fetal bovine serum and 200 mg/ μ l insulin containing Snail for 96 h. * P

Techniques Used: Cell Culture

17) Product Images from "High-Efficiency Transduction of Primary Human Hematopoietic Stem/Progenitor Cells by AAV6 Vectors: Strategies for Overcoming Donor-Variation and Implications in Genome Editing"

Article Title: High-Efficiency Transduction of Primary Human Hematopoietic Stem/Progenitor Cells by AAV6 Vectors: Strategies for Overcoming Donor-Variation and Implications in Genome Editing

Journal: Scientific Reports

doi: 10.1038/srep35495

Transduction efficiency of TM-ssAAV6 and TM-scAAV6 vectors in primary human CD34 + cells. (a) Primary human cord blood-derived CD34 + cells were either mock-transduced, or transduced at day 0 at low (0.5 × 10 6 cells/ml,) or high (1 × 10 7 cells/ml) cell density with 20,000 vgs/cell of the indicated AAV6 vectors in serum free XVIVO20 medium. Two hrs later, cells were diluted to 5 × 10 5 cells/mL and switched to the expansion medium (IMDM + FBS + SCF + IL3 + Epo+ Dexamethasone + β-estradiol + β-mercapthoethanol). EGFP expression was determined by flow cytometry at day 4 and day 10 post-transduction. (b) Following mock-transduction, or transduction of CD34 + cells as described above, cells were switched to the expansion medium for 10 days, and cultured in an erythroid differentiation medium (IMDM + BSA + Insulin + Transferrin + Epo) for an additional four days. EGFP expression was determined by flow cytometry. (c , d) Vector-transduced CD34 + cells cultured in the differentiation medium were stained with hCD36-PE and hGlycophorin A − FITC and analyzed by flow cytometry for the following: non-erythroid (CD36 − /glycoA − ), and erythroid cells (CD36 + /GlycoA + ) from day 10 to day 14.
Figure Legend Snippet: Transduction efficiency of TM-ssAAV6 and TM-scAAV6 vectors in primary human CD34 + cells. (a) Primary human cord blood-derived CD34 + cells were either mock-transduced, or transduced at day 0 at low (0.5 × 10 6 cells/ml,) or high (1 × 10 7 cells/ml) cell density with 20,000 vgs/cell of the indicated AAV6 vectors in serum free XVIVO20 medium. Two hrs later, cells were diluted to 5 × 10 5 cells/mL and switched to the expansion medium (IMDM + FBS + SCF + IL3 + Epo+ Dexamethasone + β-estradiol + β-mercapthoethanol). EGFP expression was determined by flow cytometry at day 4 and day 10 post-transduction. (b) Following mock-transduction, or transduction of CD34 + cells as described above, cells were switched to the expansion medium for 10 days, and cultured in an erythroid differentiation medium (IMDM + BSA + Insulin + Transferrin + Epo) for an additional four days. EGFP expression was determined by flow cytometry. (c , d) Vector-transduced CD34 + cells cultured in the differentiation medium were stained with hCD36-PE and hGlycophorin A − FITC and analyzed by flow cytometry for the following: non-erythroid (CD36 − /glycoA − ), and erythroid cells (CD36 + /GlycoA + ) from day 10 to day 14.

Techniques Used: Transduction, Derivative Assay, Expressing, Flow Cytometry, Cytometry, Cell Culture, Plasmid Preparation, Staining

18) Product Images from "Green tea polyphenols affect invasiveness of human gastric MKN-28 cells by inhibition of LPS or TNF-α induced Matrix Metalloproteinase-9/2"

Article Title: Green tea polyphenols affect invasiveness of human gastric MKN-28 cells by inhibition of LPS or TNF-α induced Matrix Metalloproteinase-9/2

Journal: Biochimie Open

doi: 10.1016/j.biopen.2016.10.002

Effect of the pretreatment with GTPs on Xanthine–Xanthine Oxidase induced cell cytotoxicity in MKN-28 cells. MKN-28 cells were pre-incubated with the indicated GTPs concentration in serum-free medium containing 0.1% (v/v) DMSO (vehicle) for three hours; the cells were then exposed to DMEM containing Xanthine (1 mM) and Xanthine oxidase (75 mU/ml) or with serum-free medium containing the vehicle alone for additional two hours. Control cells were incubated with DMEM in the presence (10%) or in the absence of FBS, as well as in the presence of 10 −4 M GTPs. Cell viability was determined as reported in Section 2.3 . The values represent the mean of three separate experiments performed in triplicate.
Figure Legend Snippet: Effect of the pretreatment with GTPs on Xanthine–Xanthine Oxidase induced cell cytotoxicity in MKN-28 cells. MKN-28 cells were pre-incubated with the indicated GTPs concentration in serum-free medium containing 0.1% (v/v) DMSO (vehicle) for three hours; the cells were then exposed to DMEM containing Xanthine (1 mM) and Xanthine oxidase (75 mU/ml) or with serum-free medium containing the vehicle alone for additional two hours. Control cells were incubated with DMEM in the presence (10%) or in the absence of FBS, as well as in the presence of 10 −4 M GTPs. Cell viability was determined as reported in Section 2.3 . The values represent the mean of three separate experiments performed in triplicate.

Techniques Used: Incubation, Concentration Assay

Related Articles

Modification:

Article Title: Stem cell exosomes inhibit angiogenesis and tumor growth of oral squamous cell carcinoma
Article Snippet: .. HUVEC and fibroblasts were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (15-018-CV, Corning, New York, USA) supplemented with 10% fetal bovine serum (FBS) (Lonza, Walkersville, MD USA), 1% Penicillin/Streptomycin (P/S) (Life Technologies, Carlsbad, CA, USA) and 1 mM L-glutamine (Life Technologies). .. HMEC-1 cells were cultured in endothelial cells growth media 2 (EGM-2) (cc4147, Lonza).

Article Title: Comparison among Activities and Isoflavonoids from Pueraria thunbergiana Aerial Parts and Root
Article Snippet: .. Dulbecco’s modified Eagle medium (DMEM), and fetal bovine serum (FBS) were purchased from Lonza Co. Ltd. (Basel, Switzerland). .. Antibodies for actin, COX-2, and iNOS were purchased from Cell Signaling Technology (Danvers, MA, USA).

other:

Article Title: Metabolomic Analysis Reveals Increased Aerobic Glycolysis and Amino Acid Deficit in a Cellular Model of Amyotrophic Lateral Sclerosis
Article Snippet: Materials Flasks and plates were obtained from Corning Inc. High-glucose D-MEM and fetal bovine serum (FBS) were from Lonza, and high-glucose D-MEM without phenol red, geneticin (G418 sulfate), pyruvate, l -glutamine and penicillin/streptomycin were from GIBCO, Invitrogen.

Cell Culture:

Article Title: Stem cell exosomes inhibit angiogenesis and tumor growth of oral squamous cell carcinoma
Article Snippet: .. HUVEC and fibroblasts were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (15-018-CV, Corning, New York, USA) supplemented with 10% fetal bovine serum (FBS) (Lonza, Walkersville, MD USA), 1% Penicillin/Streptomycin (P/S) (Life Technologies, Carlsbad, CA, USA) and 1 mM L-glutamine (Life Technologies). .. HMEC-1 cells were cultured in endothelial cells growth media 2 (EGM-2) (cc4147, Lonza).

Article Title: Chenodeoxycholic Acid Reduces Hypoxia Inducible Factor-1α Protein and Its Target Genes
Article Snippet: .. HepG2 cells were cultured in MEM containing non-essential amino acids and 10% fetal bovine serum (FBS) (Lonza, Basel, Switzerland) in humidified air containing 5% CO2 at 37°C. ..

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    Lonza fbs
    Tm7sf2 gene presides over an anti inflammatory loop. (a) Nuclear translocation of Nrf2 and NF-κB in WT and KO <t>MEFs.</t> MEFs, grown in DMEM plus 5%LPDS, were treated with 1 µM thapsigargin. At the indicated times, cells were collected and nuclear extracts subjected to Western blotting analyses with the indicated antibodies. Lamin B was used as loading control; (b) TNFα and HO-1 gene expression in WT and KO MEFs. MEFs, grown in DMEM plus 5%LPDS, were treated with 1 µM thapsigargin for 6 hr and subjected to real time PCR analysis. (c) TNFα expression in KO MEFs grown in <t>10%FBS</t> and treated with 1 µM thapsigargin for 6 hr and subjected to real time PCR analysis. Expression of each gene was normalized to GAPDH and reported as 2 −ΔΔCt . Relative mRNA level of WT untreated cells was assumed as 1. Results are given as mean ± s.d., (n = 7). *p
    Fbs, supplied by Lonza, used in various techniques. Bioz Stars score: 92/100, based on 573 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Lonza fbs egm 2mv medium
    Culture assay with KSL, KL, SL, and CD34 + cells. Freshly isolated KSL, KL, SL, and CD34 + cells were cultured in <t>20%FBS/EGM-2MV</t> medium on vitronectin-coated 4 well-chamber slides for 7 days. a, The cells were washed by PBS for three times and stained with DAPI. b, The numbers of adherent cells were counted and analyzed (n = 3). The assay was triplicated and demonstrated similar results.
    Fbs Egm 2mv Medium, supplied by Lonza, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Lonza cas9 expression plasmid
    <t>Cas9–HE</t> induces a different pattern of indels than Cas9
    Cas9 Expression Plasmid, supplied by Lonza, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Tm7sf2 gene presides over an anti inflammatory loop. (a) Nuclear translocation of Nrf2 and NF-κB in WT and KO MEFs. MEFs, grown in DMEM plus 5%LPDS, were treated with 1 µM thapsigargin. At the indicated times, cells were collected and nuclear extracts subjected to Western blotting analyses with the indicated antibodies. Lamin B was used as loading control; (b) TNFα and HO-1 gene expression in WT and KO MEFs. MEFs, grown in DMEM plus 5%LPDS, were treated with 1 µM thapsigargin for 6 hr and subjected to real time PCR analysis. (c) TNFα expression in KO MEFs grown in 10%FBS and treated with 1 µM thapsigargin for 6 hr and subjected to real time PCR analysis. Expression of each gene was normalized to GAPDH and reported as 2 −ΔΔCt . Relative mRNA level of WT untreated cells was assumed as 1. Results are given as mean ± s.d., (n = 7). *p

    Journal: PLoS ONE

    Article Title: A Novel Role for Tm7sf2 Gene in Regulating TNF? Expression

    doi: 10.1371/journal.pone.0068017

    Figure Lengend Snippet: Tm7sf2 gene presides over an anti inflammatory loop. (a) Nuclear translocation of Nrf2 and NF-κB in WT and KO MEFs. MEFs, grown in DMEM plus 5%LPDS, were treated with 1 µM thapsigargin. At the indicated times, cells were collected and nuclear extracts subjected to Western blotting analyses with the indicated antibodies. Lamin B was used as loading control; (b) TNFα and HO-1 gene expression in WT and KO MEFs. MEFs, grown in DMEM plus 5%LPDS, were treated with 1 µM thapsigargin for 6 hr and subjected to real time PCR analysis. (c) TNFα expression in KO MEFs grown in 10%FBS and treated with 1 µM thapsigargin for 6 hr and subjected to real time PCR analysis. Expression of each gene was normalized to GAPDH and reported as 2 −ΔΔCt . Relative mRNA level of WT untreated cells was assumed as 1. Results are given as mean ± s.d., (n = 7). *p

    Article Snippet: MEFs were maintained in DMEM containing 10% FBS (Lonza, Milan, Italy) and switched to LPDS 24 hr prior to treatments.

    Techniques: Translocation Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction

    Serum deprivation causes selective cytotoxicity, caspase 3/7 activation, and increased lactate formation in G93ASOD1 cells. a The viability of the NSC-34, WT-NSC, and G93A-NSC cell lines was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay after culture with or without 5 % fetal bovine serum (FBS) for 22 h. Percentages of the MTT conversion after culture with serum (100 %; n = 12) are shown. b Activation of caspase 3/7 of the NSC-34, WT-NSC, and G93A-NSC cell lines cultured without 5 % serum for 17 h. Values are percentages of the NSC-34 activity (100 %; n = 15). c Levels of lactate in the medium (μmol/mg of protein; n = 8) of the NSC-34, WT-NSC, and G93A-NSC cell lines cultured with or without 5 % serum for 22 h. All values are mean ± s.e.m. For each parameter, statistical significance of differences was assessed by one-way ANOVA and Tukey’s post hoc test comparing the levels of the different cell lines with or without 5 % serum (respectively ### p

    Journal: Molecular Neurobiology

    Article Title: Metabolomic Analysis Reveals Increased Aerobic Glycolysis and Amino Acid Deficit in a Cellular Model of Amyotrophic Lateral Sclerosis

    doi: 10.1007/s12035-015-9165-7

    Figure Lengend Snippet: Serum deprivation causes selective cytotoxicity, caspase 3/7 activation, and increased lactate formation in G93ASOD1 cells. a The viability of the NSC-34, WT-NSC, and G93A-NSC cell lines was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay after culture with or without 5 % fetal bovine serum (FBS) for 22 h. Percentages of the MTT conversion after culture with serum (100 %; n = 12) are shown. b Activation of caspase 3/7 of the NSC-34, WT-NSC, and G93A-NSC cell lines cultured without 5 % serum for 17 h. Values are percentages of the NSC-34 activity (100 %; n = 15). c Levels of lactate in the medium (μmol/mg of protein; n = 8) of the NSC-34, WT-NSC, and G93A-NSC cell lines cultured with or without 5 % serum for 22 h. All values are mean ± s.e.m. For each parameter, statistical significance of differences was assessed by one-way ANOVA and Tukey’s post hoc test comparing the levels of the different cell lines with or without 5 % serum (respectively ### p

    Article Snippet: Materials Flasks and plates were obtained from Corning Inc. High-glucose D-MEM and fetal bovine serum (FBS) were from Lonza, and high-glucose D-MEM without phenol red, geneticin (G418 sulfate), pyruvate, l -glutamine and penicillin/streptomycin were from GIBCO, Invitrogen.

    Techniques: Activation Assay, MTT Assay, Cell Culture, Activity Assay

    Culture assay with KSL, KL, SL, and CD34 + cells. Freshly isolated KSL, KL, SL, and CD34 + cells were cultured in 20%FBS/EGM-2MV medium on vitronectin-coated 4 well-chamber slides for 7 days. a, The cells were washed by PBS for three times and stained with DAPI. b, The numbers of adherent cells were counted and analyzed (n = 3). The assay was triplicated and demonstrated similar results.

    Journal: PLoS ONE

    Article Title: CD34+ Cells Represent Highly Functional Endothelial Progenitor Cells in Murine Bone Marrow

    doi: 10.1371/journal.pone.0020219

    Figure Lengend Snippet: Culture assay with KSL, KL, SL, and CD34 + cells. Freshly isolated KSL, KL, SL, and CD34 + cells were cultured in 20%FBS/EGM-2MV medium on vitronectin-coated 4 well-chamber slides for 7 days. a, The cells were washed by PBS for three times and stained with DAPI. b, The numbers of adherent cells were counted and analyzed (n = 3). The assay was triplicated and demonstrated similar results.

    Article Snippet: After washing with PBS, 2,000 of the DiI-labeled cells were mixed with 40,000 HUVECs in 100 µL of 10% FBS/EGM-2MV medium (Lonza) in order to evaluate the contribution of EPCs to EC-derived tube formation.

    Techniques: Isolation, Cell Culture, Staining

    Assessment of endothelial markers in cultured KSL, KL, SL, and CD34 + cells by immunocytostaining and real-time RT-PCR. Freshly isolated cells were cultured in 20%FBS/EGM-2MV medium on vitronectin-coated 4 well-chamber slides for 7 days. a, Adherent cells were stained with endothelial markers, VE-cadherin, vWF, and Flk-1. b, mRNA expression levels of the markers in 3-days cultured cells were assessed by quantitative real-time RT-PCR. The mRNA expressions were normalized to GAPDH (n = 3). All assays were triplicated and demonstrated similar results.

    Journal: PLoS ONE

    Article Title: CD34+ Cells Represent Highly Functional Endothelial Progenitor Cells in Murine Bone Marrow

    doi: 10.1371/journal.pone.0020219

    Figure Lengend Snippet: Assessment of endothelial markers in cultured KSL, KL, SL, and CD34 + cells by immunocytostaining and real-time RT-PCR. Freshly isolated cells were cultured in 20%FBS/EGM-2MV medium on vitronectin-coated 4 well-chamber slides for 7 days. a, Adherent cells were stained with endothelial markers, VE-cadherin, vWF, and Flk-1. b, mRNA expression levels of the markers in 3-days cultured cells were assessed by quantitative real-time RT-PCR. The mRNA expressions were normalized to GAPDH (n = 3). All assays were triplicated and demonstrated similar results.

    Article Snippet: After washing with PBS, 2,000 of the DiI-labeled cells were mixed with 40,000 HUVECs in 100 µL of 10% FBS/EGM-2MV medium (Lonza) in order to evaluate the contribution of EPCs to EC-derived tube formation.

    Techniques: Cell Culture, Quantitative RT-PCR, Isolation, Staining, Expressing

    Cas9–HE induces a different pattern of indels than Cas9

    Journal: Nature Communications

    Article Title: CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair

    doi: 10.1038/s41467-018-03475-7

    Figure Lengend Snippet: Cas9–HE induces a different pattern of indels than Cas9

    Article Snippet: RG37 cells were cultured in DMEM supplemented with 10% FBS and nucleofected with 1 μg of Cas9 expression plasmid, 1 μg of guide RNA expression plasmid, and 1 μg of GFP donor using NHDF solution (Lonza) and Amaxa P-022 program.

    Techniques:

    HDR stimulation by the HE domain takes place at different target genes and can depend on the guide RNA used. a . Targeted integration of the donor plasmid results in in-frame insertion of E2A - neoR . G418(neomycin)-resistant colonies were counted after Cresyl violet staining to measure HDR-mediated events and normalized by the number of colonies obtained with Cas9 to give the relative HDR frequencies indicated. Data represented are from three independent experiments for TGIF2 , RAD21 , and CREB genes and from four experiments for ATF4 and GABP . Error bars indicate standard deviation. b Relative frequencies of HDR induced by Cas9–HE were compared to those induced by Cas9 with the indicated guide RNAs, which all cleave to a small 50-bp region of the AAVS1 locus, and a common p84∆ donor plasmid, harboring ~800-bp homology arms. P84∆ was derived from the p84 donor plasmid depicted in Fig. 1a by shortening the homology arms so that they would not be cleaved by any of the guide RNAs. Asterisks indicate that the difference is statistically significant when comparing Cas9–HE to Cas9 in t -test (* P . Error bars indicate standard deviation

    Journal: Nature Communications

    Article Title: CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair

    doi: 10.1038/s41467-018-03475-7

    Figure Lengend Snippet: HDR stimulation by the HE domain takes place at different target genes and can depend on the guide RNA used. a . Targeted integration of the donor plasmid results in in-frame insertion of E2A - neoR . G418(neomycin)-resistant colonies were counted after Cresyl violet staining to measure HDR-mediated events and normalized by the number of colonies obtained with Cas9 to give the relative HDR frequencies indicated. Data represented are from three independent experiments for TGIF2 , RAD21 , and CREB genes and from four experiments for ATF4 and GABP . Error bars indicate standard deviation. b Relative frequencies of HDR induced by Cas9–HE were compared to those induced by Cas9 with the indicated guide RNAs, which all cleave to a small 50-bp region of the AAVS1 locus, and a common p84∆ donor plasmid, harboring ~800-bp homology arms. P84∆ was derived from the p84 donor plasmid depicted in Fig. 1a by shortening the homology arms so that they would not be cleaved by any of the guide RNAs. Asterisks indicate that the difference is statistically significant when comparing Cas9–HE to Cas9 in t -test (* P . Error bars indicate standard deviation

    Article Snippet: RG37 cells were cultured in DMEM supplemented with 10% FBS and nucleofected with 1 μg of Cas9 expression plasmid, 1 μg of guide RNA expression plasmid, and 1 μg of GFP donor using NHDF solution (Lonza) and Amaxa P-022 program.

    Techniques: Plasmid Preparation, Staining, Standard Deviation, Derivative Assay

    Stimulation of transgene integration by Cas9–HE and Cas9–Geminin. Relative frequencies of HDR and indels induced by Cas9 or fusion of Cas9 to HE domain, Geminin degron, or both. Human HEK293 cells were transfected with the indicated Cas9 plasmids, T2 guide RNA, and GFP transgene donor with homology arms to the AAVS1 targeted locus. HDR-mediated transgene integration was measured by FACS analysis of GFP-positive cells, resulting from targeted GFP transgene integration. Indels at the cleavage site were measured by the T7E1 assay. The results are expressed as the mean of relative HDR or indel frequency calculated by normalizing every HDR or indel frequency by that induced by Cas9. Asterisks indicate that the difference is statistically significant when comparing Cas9–HE, Cas9–HE–Geminin, and Cas9–Geminin to Cas9 in t -test (* P

    Journal: Nature Communications

    Article Title: CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair

    doi: 10.1038/s41467-018-03475-7

    Figure Lengend Snippet: Stimulation of transgene integration by Cas9–HE and Cas9–Geminin. Relative frequencies of HDR and indels induced by Cas9 or fusion of Cas9 to HE domain, Geminin degron, or both. Human HEK293 cells were transfected with the indicated Cas9 plasmids, T2 guide RNA, and GFP transgene donor with homology arms to the AAVS1 targeted locus. HDR-mediated transgene integration was measured by FACS analysis of GFP-positive cells, resulting from targeted GFP transgene integration. Indels at the cleavage site were measured by the T7E1 assay. The results are expressed as the mean of relative HDR or indel frequency calculated by normalizing every HDR or indel frequency by that induced by Cas9. Asterisks indicate that the difference is statistically significant when comparing Cas9–HE, Cas9–HE–Geminin, and Cas9–Geminin to Cas9 in t -test (* P

    Article Snippet: RG37 cells were cultured in DMEM supplemented with 10% FBS and nucleofected with 1 μg of Cas9 expression plasmid, 1 μg of guide RNA expression plasmid, and 1 μg of GFP donor using NHDF solution (Lonza) and Amaxa P-022 program.

    Techniques: Transfection, FACS