Journal: Genes

Article Title: Effect of 20(S)-Hydroxycholesterol on Multilineage Differentiation of Mesenchymal Stem Cells Isolated from Compact Bones in Chicken

doi: 10.3390/genes11111360

Figure Lengend Snippet: 20S activates osteogenic genes through the Hedgehog signaling pathway. Confluent chicken bone marrow stem cells (cBMSCs) were pretreated for 2 h with control vehicle (VEH) or 4 µM cyclopamine (CYC), followed by 24 h and 72 h of treatment with (1) control vehicle ( C ), (2) osteogenic media (OM) containing DMEM with 10% FBS, 50 µg/mL ascorbate, 0.5 µM DXA, and 10 mM β-glycerophosphate, (3) 10 µM 20S, or (4) OM + 10 µM 20S. RNA was isolated, reverse transcribed to cDNA, and qRT-PCR was conducted to analyze osteogenic gene expression. GAPDH was used as a housekeeping gene. Fold changes in gene expression relative to the control were calculated using the −ΔΔCt method. mRNA expression of ( A1 , A2 ) RunX2, ( B1 , B2 ) BMP2, ( C1 , C2 ) BSP, ( D1 , D2 ) BGLAP, ( E1 , E2 ) Gli-1, ( F1 , F2 ) Ptch, and ( G1 , G2 ) Shh were significantly reduced in cells treated with OM + 20S in the Cyc pretreated group compared to the VEH pretreated group. Bars represent mean ± SEM of triplicate determinations. Bars without a letter (a–b) in common are significantly different ( p

Article Snippet: The supernatant was discarded, and cell pellets were suspended in 10 mL DMEM containing 10% fetal bovine serum (FBS) (GE Healthcare, Chicago, IL, USA), 100 U/mL penicillin, 100 µg/mL streptomycin, and L-glutamate (Mediatech, Manassas, VA, USA).

Techniques: Isolation, Quantitative RT-PCR, Expressing

Journal: Genes

Article Title: Effect of 20(S)-Hydroxycholesterol on Multilineage Differentiation of Mesenchymal Stem Cells Isolated from Compact Bones in Chicken

doi: 10.3390/genes11111360

Figure Lengend Snippet: Mineral deposition in mesenchymal stem cells isolated from chicken compact bone stem cells treated with different levels of 20S oxysterol for 14 days (detected by Von Kossa stain). Calcium deposition was increased in cells treated with 20S oxysterol compared to control cells. Cells were treated with ( A ) Control, ( B ) osteogenic media (OM) containing DMEM with 10% FBS, 50 µg/mL ascorbate, 0.5 µM DXA, and 10 mM β-glycerophosphate, ( C ) OM + 2.5 µM 20S, ( D ) OM + 5 µM 20S, or ( E ) OM + 10 µM 20S. Pictures were taken by Olympus DP70 at 20X.

Article Snippet: The supernatant was discarded, and cell pellets were suspended in 10 mL DMEM containing 10% fetal bovine serum (FBS) (GE Healthcare, Chicago, IL, USA), 100 U/mL penicillin, 100 µg/mL streptomycin, and L-glutamate (Mediatech, Manassas, VA, USA).

Techniques: Isolation, Staining

Journal: Genes

Article Title: Effect of 20(S)-Hydroxycholesterol on Multilineage Differentiation of Mesenchymal Stem Cells Isolated from Compact Bones in Chicken

doi: 10.3390/genes11111360

Figure Lengend Snippet: Osteogenic effects of 20S oxysterol in mesenchymal stem cells. Relative levels of mRNA expression of osteogenic genes ( A ) BSP, ( B ) BMP2, ( C ) Gli-1, ( D ) Col1A2, ( E ) Ptch, ( F ) BGLAP, ( G ) Shh, and ( H ) RunX2 in chicken compact bone-derived mesenchymal stem cells (cBMSCs) treated with different levels of oxysterol. cBMSCs were treated with (1) Control (C), (2) osteogenic media (OM) containing DMEM with 10% FBS, 50 µg/mL ascorbate, 0.5 µM DXA, and 10 mM β-glycerophosphate, (3) OM + 2.5 µM 20S, (4) OM + 5 µM 20S, or (5) OM + 10 µM 20S. Cells were harvested at 24 h, 72 h, and 7 d after treatment. RNA was isolated, reverse transcribed to cDNA, and qRT-PCR was conducted to analyze osteogenic gene expression. GAPDH was used as a housekeeping gene. Fold changes in gene expression relative to the control were calculated using the −ΔΔCt method. Bars represent mean ± SEM for triplicate determinations. Within each harvested period, bars without a letter (a–b) in common are significantly different ( p

Article Snippet: The supernatant was discarded, and cell pellets were suspended in 10 mL DMEM containing 10% fetal bovine serum (FBS) (GE Healthcare, Chicago, IL, USA), 100 U/mL penicillin, 100 µg/mL streptomycin, and L-glutamate (Mediatech, Manassas, VA, USA).

Techniques: Expressing, Derivative Assay, Isolation, Quantitative RT-PCR

Journal: Genes

Article Title: Effect of 20(S)-Hydroxycholesterol on Multilineage Differentiation of Mesenchymal Stem Cells Isolated from Compact Bones in Chicken

doi: 10.3390/genes11111360

Figure Lengend Snippet: Mineral deposition in mesenchymal stem cells isolated from chicken compact bone stem cells treated with different levels of 20S oxysterol for 14 days (detected by alizarin red test). Calcium deposition was increased in cells treated with 20S oxysterol compared to control cells. Cells were treated with ( A ) Control ( B ) osteogenic media (OM) containing DMEM with 10% FBS, 50 µg/mL ascorbate, 0.5 µM DXA, and 10 mM β-glycerophosphate, ( C ) OM + 2.5 µM 20S, ( D ) OM + 5 µM 20S, or ( E ) OM + 10 µM 20S. Pictures were taken by Olympus DP70 at 4X.

Article Snippet: The supernatant was discarded, and cell pellets were suspended in 10 mL DMEM containing 10% fetal bovine serum (FBS) (GE Healthcare, Chicago, IL, USA), 100 U/mL penicillin, 100 µg/mL streptomycin, and L-glutamate (Mediatech, Manassas, VA, USA).

Techniques: Isolation

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Acylcarnitines activate proinflammatory signaling pathways

doi: 10.1152/ajpendo.00656.2013

Figure Lengend Snippet: l -C14 carnitine does not induce NFκB in TLR 2/1 or 2/6 in transfected HEK-293T cells. HEK-293T cells were cultured in 10% FBS/DMEM medium and cotransfected with TLR2 and TLR1, TLR2 and TLR6 in addition to NF-κB-luciferase reporter and

Article Snippet: These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) FBS (premium select FBS, cat. no. S11595, lot no. K0109; Atlanta Biologicals, Lawrenceville, GA), 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Transfection, Cell Culture, Luciferase

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Acylcarnitines activate proinflammatory signaling pathways

doi: 10.1152/ajpendo.00656.2013

Figure Lengend Snippet: l -C14 carnitine induced reactive oxygen species (ROS) but not mitochondrial ROS in murine monocyte/macrophages. RAW 264.7 cells were serum-starved for 6 h (0.25% FBS/DMEM) then treated with LPS (100 ng/mL), 25 μM l -C14 acylcarnitine ( l -C14 carn),

Article Snippet: These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) FBS (premium select FBS, cat. no. S11595, lot no. K0109; Atlanta Biologicals, Lawrenceville, GA), 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques:

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Acylcarnitines activate proinflammatory signaling pathways

doi: 10.1152/ajpendo.00656.2013

Figure Lengend Snippet: Induction of COX-2 and proinflammatory cytokines are reduced in MyD88 knock-down RAW 264.7 cells. Stable MyD88 KD cells were generated by lentiviral mediated shRNA in RAW 264.7 cells. Cells were then serum-starved for 4–6 h (0.25% FBS/DMEM) then

Article Snippet: These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) FBS (premium select FBS, cat. no. S11595, lot no. K0109; Atlanta Biologicals, Lawrenceville, GA), 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Generated, shRNA

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Acylcarnitines activate proinflammatory signaling pathways

doi: 10.1152/ajpendo.00656.2013

Figure Lengend Snippet: l -C14 carnitine increases phosphorylation of ERK and JNK in a time-dependent manner: MAP kinases and NF-κB contribution to l -C14 carnitine induced cytokine production. RAW 264.7 cells were serum-starved for 6 h (0.25% FBS/DMEM) then treated with

Article Snippet: These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) FBS (premium select FBS, cat. no. S11595, lot no. K0109; Atlanta Biologicals, Lawrenceville, GA), 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques:

Journal: Anti-cancer drugs

Article Title: Effect of artemisinin derivatives on apoptosis and cell cycle in prostate cancer cells

doi: 10.1097/CAD.0b013e328336f57b

Figure Lengend Snippet: Effect of 2Py on survivin, Bcl-2, and Bax in LNCaP, C4-2, PC-3 and DU 145 cells Cells were brought to 50% confluence and then cultured in RPMI 1640 with 10% FBS medium with DMSO or 15 µM 2Py for 48 h, cell lysates were isolated and western blotting analysis performed using antibodies to survivin (n=4), Bax (n=3), and Bcl-2 (n=3), with β-actin as control.

Article Snippet: After treatment in RPMI 1640 with 10% fetal bovine serum (FBS) (Atlanta Biologicals) with DMSO or 15 µM 2Py for 48 h, cells were fixed and incubated with rabbit polyclonal anti-human β-catenin antibody (5 µg/mL Santa Cruz Biotechnology Inc. Santa Cruz, CA) or control rabbit IgG (5 µg/mL Vector laboratories Inc. Burlingame, CA) and then with goat-anti-rabbit Alexa Fluor 488 at a dilution of 1:400 respectively, and mounted with ProlongGold anti-fade reagent w/DAPI (Invitrogen Corp.).

Techniques: Cell Culture, Isolation, Western Blot

Journal: Anti-cancer drugs

Article Title: Effect of artemisinin derivatives on apoptosis and cell cycle in prostate cancer cells

doi: 10.1097/CAD.0b013e328336f57b

Figure Lengend Snippet: Cell number as assessed by crystal violet assay in LNCaP, C4-2 and PC-3 cells Cells were brought to 50% confluence and then cultured in RPMI 1640 with 10% FBS medium with DMSO, 10, or 25 µM DHA, ON-2Py or 2Py for 24, 48, and 72 h. Relative cell number was assessed by crystal violet assay (n = 4). Results are expressed as the mean ± SD. * indicates significant difference from control (p

Article Snippet: After treatment in RPMI 1640 with 10% fetal bovine serum (FBS) (Atlanta Biologicals) with DMSO or 15 µM 2Py for 48 h, cells were fixed and incubated with rabbit polyclonal anti-human β-catenin antibody (5 µg/mL Santa Cruz Biotechnology Inc. Santa Cruz, CA) or control rabbit IgG (5 µg/mL Vector laboratories Inc. Burlingame, CA) and then with goat-anti-rabbit Alexa Fluor 488 at a dilution of 1:400 respectively, and mounted with ProlongGold anti-fade reagent w/DAPI (Invitrogen Corp.).

Techniques: Crystal Violet Assay, Cell Culture

Journal: Anti-cancer drugs

Article Title: Effect of artemisinin derivatives on apoptosis and cell cycle in prostate cancer cells

doi: 10.1097/CAD.0b013e328336f57b

Figure Lengend Snippet: Effect of 2Py on apoptosis and cell cycle in LNCaP, C4-2, PC-3 and DU 145 cells Cells were brought to 50% confluence and then cultured in RPMI 1640 with 10% FBS medium with DMSO or 15 µM 2Py for 48 h. The cells were trypsinized and stained with propidium iodide and analyzed on a flow cytometer. The percentage of apoptotic events (Panel A), cell viability (Panel B), and the percentage of cells in G0 and G2M and S phase of the cell cycle (Panels C, D and E) were determined (n=3). Histograms of cell cycle distribution in control and 2Py treated cells (Panel F).

Article Snippet: After treatment in RPMI 1640 with 10% fetal bovine serum (FBS) (Atlanta Biologicals) with DMSO or 15 µM 2Py for 48 h, cells were fixed and incubated with rabbit polyclonal anti-human β-catenin antibody (5 µg/mL Santa Cruz Biotechnology Inc. Santa Cruz, CA) or control rabbit IgG (5 µg/mL Vector laboratories Inc. Burlingame, CA) and then with goat-anti-rabbit Alexa Fluor 488 at a dilution of 1:400 respectively, and mounted with ProlongGold anti-fade reagent w/DAPI (Invitrogen Corp.).

Techniques: Cell Culture, Staining, Flow Cytometry, Cytometry

Journal: Anti-cancer drugs

Article Title: Effect of artemisinin derivatives on apoptosis and cell cycle in prostate cancer cells

doi: 10.1097/CAD.0b013e328336f57b

Figure Lengend Snippet: Effect of 2Py on C-Myc, Cyclin D1, AR, PSA, and β-catenin expression in LNCaP and C4-2 cells (A) Cells were brought to 50% confluence and then cultured in RPMI 1640 with 10% FBS medium with DMSO or 15 µM 2Py for 48 h, cell lysates were isolated and Western blotting analysis performed using antibodies to Cyclin D1 (n=3), c-Myc (n=3), AR (n=3), PSA (n=3), and β-catenin (n=2) with β-actin as control (n=3). (B) Immunocytochemical analysis of β-catenin in LNCaP and C4-2 cells cultured in 10% FBS medium with DMSO or 15 µM 2Py for 24 h. Arrows indicate cytoplasmic relocalization of β-catenin after 2Py treatment.

Article Snippet: After treatment in RPMI 1640 with 10% fetal bovine serum (FBS) (Atlanta Biologicals) with DMSO or 15 µM 2Py for 48 h, cells were fixed and incubated with rabbit polyclonal anti-human β-catenin antibody (5 µg/mL Santa Cruz Biotechnology Inc. Santa Cruz, CA) or control rabbit IgG (5 µg/mL Vector laboratories Inc. Burlingame, CA) and then with goat-anti-rabbit Alexa Fluor 488 at a dilution of 1:400 respectively, and mounted with ProlongGold anti-fade reagent w/DAPI (Invitrogen Corp.).

Techniques: Expressing, Cell Culture, Isolation, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Angiotensin II activates AMPK for execution of apoptosis through energy-dependent and -independent mechanisms

doi: 10.1152/ajplung.00072.2011

Figure Lengend Snippet: Src-homology two-domain-containing phosphatase-2 (SHP-2) activation by Ang II requires AMPK. PAEC were grown to 80% confluence and placed in 0.1% FBS/RPMI overnight. A and B : PAEC were treated with Ang II (10 μM) for the indicated times. Cells were pretreated with or without the AMPK inhibitor compound C (40 μM) for 20 min. A : equal amounts of protein from cell lysates were immunoprecipitated with anti-SHP-2. Immunoprecipitated SHP-2 protein was then subjected to phosphatase assays. B : equal amounts of protein were used for SHP-2 immunoprecipitation and blotted for tyrosine phosphorylation. Lower panel shows densitometry of phospho-SHP-2 bands. C : PAEC were treated with 10 μM Ang II for 5 min before preparation of cell lysates. Equal amounts of protein were used for SHP-2 immunoprecipation. Immunoprecipitated protein was then Western blotted for phospho-AMPK ( top ) or for SHP-2 ( bottom ). Lower bar graph indicates phospho-AMPK band density normalized to SHP-2 band density. All data show means ± SD. *Indicates statistical significance from control, P

Article Snippet: Passage 2–8 cells were used for all experiments and were cultured in RPMI 1640 medium containing 10% FBS (Gemini Bioproducts, Woodland, CA), 1% penicillin/streptomycin, and 0.5% fungizone (Invitrogen, Carlsbad, CA).

Techniques: Activation Assay, Immunoprecipitation, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Angiotensin II activates AMPK for execution of apoptosis through energy-dependent and -independent mechanisms

doi: 10.1152/ajplung.00072.2011

Figure Lengend Snippet: Increase of ATP production induced by angiotensin type 2 receptor (AT 2 ) stimulation. Pulmonary artery endothelial cells (PAEC) were grown to 80% confluence and placed in 0.1% FBS/RPMI overnight before treatment with angiotensin II (Ang II) (10 μM). Relative ATP levels were measured. A : Ang II was added for the indicated times. B : Ang II-induced ATP productions were assayed after 2 h in the presence and absence of compound C (40 μM) or oligomycin (50 nM). Compound C or oligomycin was added 20 min before Ang II. C : Ang II AT 2 receptor antagonist PD123319 (50 μM) was added 20 min before the addition of Ang II for 2 h. All data show means ± SD. * P

Article Snippet: Passage 2–8 cells were used for all experiments and were cultured in RPMI 1640 medium containing 10% FBS (Gemini Bioproducts, Woodland, CA), 1% penicillin/streptomycin, and 0.5% fungizone (Invitrogen, Carlsbad, CA).

Techniques:

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Angiotensin II activates AMPK for execution of apoptosis through energy-dependent and -independent mechanisms

doi: 10.1152/ajplung.00072.2011

Figure Lengend Snippet: ATP generation and AMPK are required for Ang II-induced apoptosis in PAEC. A and B : PAEC were grown to 80% confluence and placed in 0.1% FBS/RPMI overnight. Neutral comet assays were performed to detect apoptotic cells. Percentage of apoptotic cells were then determined. A : cells were treated with oligomycin (50 nM) for 1 h in glucose-free medium (RPMI, 0% FBS). Cells were washed twice in 10% FBS/RPMI before the addition of Ang II (10 μM) for 16 h. B : cells were treated with and without the AMPK inhibitor compound C (40 μM). C : mitochondria-free cell lysates were Western blotted for active cytochrome c and caspase 3. Blots were stripped and probed for β-actin as a loading control. Representative results are shown. Bar graphs represent normalized band densities for both cytochrome c and activated caspase 3. All bar graphs show means ± SD. * P

Article Snippet: Passage 2–8 cells were used for all experiments and were cultured in RPMI 1640 medium containing 10% FBS (Gemini Bioproducts, Woodland, CA), 1% penicillin/streptomycin, and 0.5% fungizone (Invitrogen, Carlsbad, CA).

Techniques: Western Blot

Journal: Arthritis Research & Therapy

Article Title: Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

doi: 10.1186/s13075-018-1699-4

Figure Lengend Snippet: Arhalofenate acid activated AMPK downstream targets involved in regulation of mitochondrial function and maintained mitochondrial cristae area. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before stimulation with monosodium urate (MSU) crystals (0.2 mg/mL) for 1 h or 18 h in RPMI containing 1% FBS. Cell lysates prepared from 18-h treatment samples were subjected to Western blot analysis of phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α, expression of sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), and mitochondrial transcription factor A (TFAM), and expression of thioredoxin (TRX)1, TRX2, and thioredoxin-interacting protein (TXNIP) ( a ). Cells from 1-h treatment were then stained with MitoSOX Red and MitoTracker Green and visualized by fluorescence microscopy ( b , magnification 20×). TEM analysis was performed to examine mitochondrial cristae (the folds of inner mitochondrial membrane indicated by white arrows in c ), and the cristae volume density are presented ( d ). Data in a and b are representative of three individual experiments. Data in c are representative of 30 cells examined for each condition. Data in d are the mean ± SEM of 30 cells. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid

Article Snippet: Briefly, bone marrow cells were cultured in complete RPMI media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) in the presence of macrophage colony-stimulating factor (M-CSF; 20 ng/mL; Gemini Bio-products, West Sacramento, CA).

Techniques: Western Blot, Expressing, Pyrolysis Gas Chromatography, Staining, Fluorescence, Microscopy, Transmission Electron Microscopy

Journal: Arthritis Research & Therapy

Article Title: Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

doi: 10.1186/s13075-018-1699-4

Figure Lengend Snippet: Arhalofenate acid prevented prolonged accumulation of p62 by promoting autophagy flux in response to MSU crystals. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) for 2 h in the presence or absence of bafilomycin (Baf; 100 nM) and for 6 h in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis of LC3 and p62 ( a ). TEM was performed to examine autophagosomes (indicated by black arrows in b ), and the numbers of autophagosomes containing electron dense material per μm 2 are presented ( c ). Data in a and b are representative of three individual experiments. Data in c are the mean ± SEM of 30 cells. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid

Article Snippet: Briefly, bone marrow cells were cultured in complete RPMI media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) in the presence of macrophage colony-stimulating factor (M-CSF; 20 ng/mL; Gemini Bio-products, West Sacramento, CA).

Techniques: Western Blot, Transmission Electron Microscopy

Journal: Arthritis Research & Therapy

Article Title: Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

doi: 10.1186/s13075-018-1699-4

Figure Lengend Snippet: Arhalofenate acid attenuates MSU crystal-induced IL-1β release by inhibiting NLRP3 inflammasome activation in BMDMs in vitro. BMDMs were pretreated with arhalofenate acid at a concentration of 100 μM for 1 h before being stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) in RPMI containing 1% FBS for 18 h. The conditioned media was used for ELISA for interleukin (IL)-1β ( a ), and the cell lysates were subjected to Western blot analysis ( b ) for expression of NLRP3, pro-caspase 1, and cleaved caspase 1 (p10). Data in a are the mean ± SD of three individual experiments, and p values represent comparisons between none and MSU crystals alone, or between MSU crystal alone and MSU crystals plus arhalofenate acid. Data in b are representative of three individual experiments

Article Snippet: Briefly, bone marrow cells were cultured in complete RPMI media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) in the presence of macrophage colony-stimulating factor (M-CSF; 20 ng/mL; Gemini Bio-products, West Sacramento, CA).

Techniques: Activation Assay, In Vitro, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Arthritis Research & Therapy

Article Title: Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

doi: 10.1186/s13075-018-1699-4

Figure Lengend Snippet: Arhalofenate acid inhibited MSU crystal-induced IL-1β via AMPK in BMDMs in vitro. BMDMs were treated with arhalofenate acid at 100 μM for 1 h before being stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) for 18 h in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis for phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α ( a ). The conditioned medium was used for ELISA analysis of interleukin (IL)-1β release ( b ). Data in a are representative of three individual experiments. Data in b are the mean ± SD of three individual experiments. The p values in b represent comparisons between none and MSU crystals alone in the presence or absence of arhalofenate acid in either wild-type (WT) or AMPKα1 knockout (KO) BMDMs. ns not significant

Article Snippet: Briefly, bone marrow cells were cultured in complete RPMI media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) in the presence of macrophage colony-stimulating factor (M-CSF; 20 ng/mL; Gemini Bio-products, West Sacramento, CA).

Techniques: In Vitro, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: Arthritis Research & Therapy

Article Title: Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

doi: 10.1186/s13075-018-1699-4

Figure Lengend Snippet: Arhalofenate acid induced phosphorylation of AMPKα and expression of SIRT1 in BMDMs in vitro. BMDMs prepared from wild-type (WT) and AMPKα1 knockout (KO) mice were pretreated with arhalofenate acid at the concentrations indicated for 18 h ( a ) or at 100 μM for 1 h ( b ) in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis for phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α and sirtuin 1 (SIRT1). Data in both a and b are representative of three individual experiments

Article Snippet: Briefly, bone marrow cells were cultured in complete RPMI media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) in the presence of macrophage colony-stimulating factor (M-CSF; 20 ng/mL; Gemini Bio-products, West Sacramento, CA).

Techniques: Expressing, In Vitro, Knock-Out, Mouse Assay, Western Blot

Journal: PLoS ONE

Article Title: Prospective Identification of Glioblastoma Cells Generating Dormant Tumors

doi: 10.1371/journal.pone.0044395

Figure Lengend Snippet: Angiogenic potential comparison between cells of U-87 MG and Clone #1. A. C.M. of U-87 MG cells induced extensive sprouting of endothelial cells (upper panel) from aortic rings resected from mice as compared with C.M. of Clone #1 (lower panel). B. HUVEC migrate towards C.M. of U-87 MG cells (red bar) at a significantly increased rate ( p = 1.6×10 −13 ) compared with that of Clone #1 (black bar). DMEM containing 10% FBS served as positive control; DMEM served as negative control. C. HUVEC’s ability to form capillary-like tube structures on Matrigel® is significantly higher ( p = 0.001) in the presence of C.M. of U-87 MG cells (red bar) compared with that in the presence of C.M. of Clone #1 cells (black bar). D. Contrast-enhanced ultrasound imaging of subcutaneously-inoculated plugs containing C.M. media from U-87 MG cells (red bar, n = 3) showed increased vascularization compared with C.M. from Clone #1 cells (black bar, n = 3) ( p = 0.04). Data represent mean± s.d. from three independent experiments. * p

Article Snippet: Materials Dulbecco’s modified Eagle’s medium (DMEM), Fetal Bovine Serum (FBS), Penicillin, Streptomycin, Nystatin, and L-glutamine were purchased from Biological Industries (Kibbutz Beit Haemek, Israel).

Techniques: Mouse Assay, Positive Control, Negative Control, Imaging

Journal: PLoS ONE

Article Title: Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

doi: 10.1371/journal.pone.0004417

Figure Lengend Snippet: Effect of trypsin on NLP formation. NLP were prepared by adding 1 mM of CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 to DMEM containing the indicated amounts of serum, in the presence or absence of 0.5% trypsin, followed by incubation at 37°C for 1 day (A–D) or 3 days (E–H). Trypsin was shown not only to release the inhibition caused by FBS or HS, but also to produce a dose-dependent increase in the amount of precipitation that increased with the serum dosage. (I) Trypsin treatment on NLP as demonstrated by SDS-PAGE. NLP formed as in Fig. 9 from 3 mM each of the 3 precipitating reagents as well as 1% FBS in DMEM, either in the absence (lane 1) or presence (lane 2) of 0.5% trypsin. After overnight incubation of the 1 ml incubation mixture at 37°C, 100 µl was removed, pelleted by centrifugation, and washed twice in DMEM, and resuspended in 16 µl of 50 mM EDTA in double-distilled water for SDS-PAGE. Lane 3 contains 5 µg of trypsin, as control, which shows as a 25 kDa band. Note a prominent 72 kDa band and two fainter 54 kDa and 30 kDa bands associated with FBS NLP (lane 1), all of which disappears with trypsin treatment (lane 2).

Article Snippet: All strains of NB were cultured as described earlier , using DMEM with or without FBS (Biological Industries, Kibbutz Beit Haemek, Isreal ; PAA Laboratories, Pashing, Austria).

Techniques: Incubation, Inhibition, SDS Page, Centrifugation

Journal: PLoS ONE

Article Title: Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

doi: 10.1371/journal.pone.0004417

Figure Lengend Snippet: Energy-dispersive X-ray spectroscopy of NLP and NB. EDX results were selected to illustrate the wide spectrum of NLP compositions, with respect to the amounts of calcium and phosphorus incorporated, and their similarities to NB. (A) Elemental composition of NLP prepared by adding CaCl 2 and (NH 4 ) 2 CO 3 at 50 mM into DMEM corresponding to calcium carbonate. (B and C) Amorphous CaCO 3 NLP produced as in (A) except that the precipitating reagents were added to DMEM and incubated at room temperature for 2 days (B) or one month (C), showing increased incorporation of P with prolonged incubation. Ca/P ratios are 5.09 for (B) and 1.60 for (C), with the latter close to the theoretical value of 1.67 associated with stoichiometric HAP. (D–H) Formation of NLP with various inputs of ions, including P, yielding different Ca/P ratios. (D) NLP formed from 0.25 M each of CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 , mixed in with DMEM at vol. ratios of 2∶5∶5∶13 that had been incubated in DMEM for 2 days, revealing the presence of magnesium ions and a Ca/P ratio of 0.78. (E–H) NLP were prepared like in (D) at vol. ratios of (E) 1∶1∶1∶22, yielding a Ca/P ratio of 1.29; (F) 3∶2∶2∶43, Ca/P ratio of 1.49; (G) 9∶5∶5∶106, Ca/P ratio of 1.76; (H) 2∶1∶1∶21, Ca/P ratio of 2.03. NLP were submitted to EDX immediately after formation. (I–L) Elemental composition of various NB preparations. (I and J) NB obtained from one-month-old DMEM cultures containing (I) 10% HS or (J) 10% FBS. Ca/P ratios: 1.75 (I) and 1.53 (J). (K) NB strain DSM 5819, after one week in DMEM without serum, giving a Ca/P ratio of 1.45. (L) “Nanons” after one week in DMEM; Ca/P ratio: 1.66.

Article Snippet: All strains of NB were cultured as described earlier , using DMEM with or without FBS (Biological Industries, Kibbutz Beit Haemek, Isreal ; PAA Laboratories, Pashing, Austria).

Techniques: Spectroscopy, Produced, Incubation

Journal: PLoS ONE

Article Title: Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

doi: 10.1371/journal.pone.0004417

Figure Lengend Snippet: Effect of serum, temperature, and EDTA on NLP formation. NLP were prepared from 3 mM of CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 in DMEM containing different amounts of FBS or HS, as indicated, followed by incubation at 37°C for 1 day (A and B), 1 week (E and F), or 4°C for 2 weeks (C and D). Note that FBS had a stronger, dose-dependent inhibitory effect on NLP formation in all 3 situations when compared with HS. Inhibition could be seen for 2 weeks at 4°C, but at 37°C, it was overcome after 1 week, and this release of inhibition was more evident with HS. (G ) NLP were prepared exactly as in Fig. 1B , by adding solutions of 0.25 M CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 (pH 7.4) to DMEM to the indicated concentrations in the presence of 10 mM EDTA, followed by incubation for 1 day. Addition of EDTA into the precipitation mix decreased the amount of NLP, except for well 6, containing 10 mM of precipitating reagents, indicating that excess EDTA is needed for the inhibition to work.

Article Snippet: All strains of NB were cultured as described earlier , using DMEM with or without FBS (Biological Industries, Kibbutz Beit Haemek, Isreal ; PAA Laboratories, Pashing, Austria).

Techniques: Incubation, Inhibition

Journal: PLoS ONE

Article Title: Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

doi: 10.1371/journal.pone.0004417

Figure Lengend Snippet: Protein profiles of NLP and NB as determined by their passage through serum-free or serum-containing medium. (A) Protein profile of HS NB maintained in DMEM containing 5% HS (lane 1) followed by transfer to serum-free DMEM and incubation for 1 day (lane 2), 4 days (lane 3), and 6 days (lane 4), after which proteins were analyzed by SDS-PAGE. A gradual disappearance of proteins bands is seen with increased incubation time in serum-free medium. (B) Protein profile of NB strain DSM 5821 after 1 day in DMEM containing 2% FBS (lane 1), followed by transfer to serum-free DMEM for 1 day (lane 2), 4 days (lane 3), and 6 days (lane 4), again showing a gradual disappearance of bands. (C) Protein profiles of NB obtained from 10% HS as in Fig. 3 (lane 1) and HS NLP formed as in Fig. 3 except that 5% HS was present in the precipitating mixture (lane 2). HS NLP were then transferred to DMEM containing 5% HS and incubated for the following periods of time: 1 hr (lane 3), 1 day (lane 4), 4 days (lane 5), 10 days (lane 6), and 14 days (lane 7). A gradual increase of bands of low molecular weight could be seen along with a fading of the high molecular weight bands, especially the 70–72 kDa band. (D) Protein profiles of NB strain DSM 5820 maintained in 10% γ-irradiated FBS (lane 1) and FBS NLP obtained as in Fig. 3 , except that 5% FBS was added to the precipitating mixture (lane 2). FBS NLP were inoculated into DMEM containing 5% FBS and incubated for the following periods of time: 2 hr (lane 3), 1 day (lane 4), 4 days (lane 5), 10 days (lane 6), and 14 days (lane 7). By day 14, the protein profile of these FBS NLP, with an increase in the number of bands and a loss of the 70–72 kDa band, closely resembled that of DSM 5820 (lane 1).

Article Snippet: All strains of NB were cultured as described earlier , using DMEM with or without FBS (Biological Industries, Kibbutz Beit Haemek, Isreal ; PAA Laboratories, Pashing, Austria).

Techniques: Incubation, SDS Page, Molecular Weight, Irradiation

Journal: PLoS ONE

Article Title: Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

doi: 10.1371/journal.pone.0004417

Figure Lengend Snippet: Protein profiles of NLP and NB cultured from serum. (A) Protein profiles of (A) NB cultured from HS and DMEM, as in Fig. 3 ; (B) NB strain DSM 5819 subcultured in serum-free DMEM for 2 days; (C) FBS NLP prepared as in Fig. 3 in the presence of 5% FBS; (D) HS NLP prepared as in Fig. 3 in the presence of 5% HS. (E) NB as in (A) after three 2-day serial passages through serum-free DMEM. (F) Lane 1: “Nanons” after two 2-day passages in serum-free DMEM. Lane 2: “Nanons” after two 2-day passages in DMEM containing 10% FBS. Gels were stained with Coomassie blue. The protein samples loaded onto each lane and their preparation are described in the Materials and Methods .

Article Snippet: All strains of NB were cultured as described earlier , using DMEM with or without FBS (Biological Industries, Kibbutz Beit Haemek, Isreal ; PAA Laboratories, Pashing, Austria).

Techniques: Cell Culture, Staining

Journal: PLoS ONE

Article Title: Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

doi: 10.1371/journal.pone.0004417

Figure Lengend Snippet: Culture of NB from serum and formation of NLP. (A) Culture of NB from FBS (top panel) and HS (bottom panel). Serum was inoculated into DMEM to the indicated concentrations. After incubating for 1 month at 37°C, NB were detected by visual inspection and A 650 (insets). For FBS, the amount of NB was maximal at 0.3%, decreasing thereafter with higher FBS concentrations, while for HS, NB were only visually noticeable at HS concentrations exceeding 3%. (B) Formation of NLP in DMEM. NLP were prepared by successively adding solutions of 0.25 M CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 (pH 7.4) into DMEM as indicated and incubated at 37°C overnight, which resulted in dose-dependent formation of NLP (top row). The same experiment was repeated in the presence of 1% FBS, which showed inhibition of NLP formation (bottom row). This serum inhibition was overcome by increased concentrations of precipitating reagents, at 3 mM and above.

Article Snippet: All strains of NB were cultured as described earlier , using DMEM with or without FBS (Biological Industries, Kibbutz Beit Haemek, Isreal ; PAA Laboratories, Pashing, Austria).

Techniques: Incubation, Inhibition

Journal: PLoS ONE

Article Title: Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

doi: 10.1371/journal.pone.0004417

Figure Lengend Snippet: Protein identification in NLP prepared from FBS and HS. Protein profiles correspond to Figs. 11C and D , reproduced here to illustrate the protein identification procedure used as well as the results obtained. NLP were prepared from DMEM containing 5% FBS or 5% HS that had been precipitated with 10 mM each of CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 . Following SDS-PAGE and Coomassie blue staining, the bands were excised (white dots) and submitted to identification by MALDI-TOF mass fingerprint analysis. The proteins identified are given along with their positions. Abbreviations used: BSA, bovine serum albumin; BSF, bovine serum fetuin-A; b Apo-A1, bovine apolipoprotein A1; HSA, human serum albumin; HSF, human serum fetuin-A; and h Apo-A1, human apolipoprotein A1. Other minor protein bands include a bovine 95 kDa band (▪) which was identified as bovine serotransferrin precursor (2 out of 2 trials, or 2/2), and other minor human bands at 165 kDa (•), 130 kDa (♦), 95 kDa (◃) identified as human complement C3 (2/2), human antithrombin (2/2), and human complement C3 (2/2), respectively.

Article Snippet: All strains of NB were cultured as described earlier , using DMEM with or without FBS (Biological Industries, Kibbutz Beit Haemek, Isreal ; PAA Laboratories, Pashing, Austria).

Techniques: SDS Page, Staining

Journal: Blood research

Article Title: Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

doi: 10.5045/br.2020.55.1.35

Figure Lengend Snippet: Count of Vero cells cultured in 10% FBS and 10% HPL. Data are expressed as means±SD of three independent experiments ( P > 0.05).

Article Snippet: Measurement of growth factors in HPL and FBS samples For HPL and FBS (#S1810-100, Biowest, Riverside, MO, USA) samples, the concentrations of IGF-1 and TGF-β1 were analyzed using enzyme-linked immunosorbent assay (ELISA) kits #IGF31-K01 (Eagle Biosciences, Inc., Amherst, NH, USA) and #SK00033-02 (Aviscera Bioscience Inc., Santa Clara, CA, USA), respectively.

Techniques: Cell Culture

Journal: Blood research

Article Title: Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

doi: 10.5045/br.2020.55.1.35

Figure Lengend Snippet: Growth factor contents in FBS and HPL samples. IGF-1 (A) , TGF-β (B) , VEGF (C) , EGF (D) , FGF (E) , PDGF (F) . Values are expressed as means±SD (N=3). a) Significantly different from FBS samples at P

Article Snippet: Measurement of growth factors in HPL and FBS samples For HPL and FBS (#S1810-100, Biowest, Riverside, MO, USA) samples, the concentrations of IGF-1 and TGF-β1 were analyzed using enzyme-linked immunosorbent assay (ELISA) kits #IGF31-K01 (Eagle Biosciences, Inc., Amherst, NH, USA) and #SK00033-02 (Aviscera Bioscience Inc., Santa Clara, CA, USA), respectively.

Techniques:

Journal: Blood research

Article Title: Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

doi: 10.5045/br.2020.55.1.35

Figure Lengend Snippet: Growth factor stability in HPL and FBS samples stored over 15 months. VEGF (A) , IGF-1 (B) , TGF-β (C) . Values are expressed as means±SD (N=3).

Article Snippet: Measurement of growth factors in HPL and FBS samples For HPL and FBS (#S1810-100, Biowest, Riverside, MO, USA) samples, the concentrations of IGF-1 and TGF-β1 were analyzed using enzyme-linked immunosorbent assay (ELISA) kits #IGF31-K01 (Eagle Biosciences, Inc., Amherst, NH, USA) and #SK00033-02 (Aviscera Bioscience Inc., Santa Clara, CA, USA), respectively.

Techniques:

Journal: Blood research

Article Title: Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

doi: 10.5045/br.2020.55.1.35

Figure Lengend Snippet: (A) MTT reduction curve for Vero cells cultured in the presence of 10% FBS, 10% HPL, and 5% HPL and in SFM. Data are expressed as means of three independent experiments ( P

Article Snippet: Measurement of growth factors in HPL and FBS samples For HPL and FBS (#S1810-100, Biowest, Riverside, MO, USA) samples, the concentrations of IGF-1 and TGF-β1 were analyzed using enzyme-linked immunosorbent assay (ELISA) kits #IGF31-K01 (Eagle Biosciences, Inc., Amherst, NH, USA) and #SK00033-02 (Aviscera Bioscience Inc., Santa Clara, CA, USA), respectively.

Techniques: MTT Assay, Cell Culture

Journal: Blood research

Article Title: Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

doi: 10.5045/br.2020.55.1.35

Figure Lengend Snippet: Count of Hep-2 cells cultivated in 10% FBS and 10% HPL. Data are expressed as means±SD of three independent experiments ( P > 0.05).

Article Snippet: Measurement of growth factors in HPL and FBS samples For HPL and FBS (#S1810-100, Biowest, Riverside, MO, USA) samples, the concentrations of IGF-1 and TGF-β1 were analyzed using enzyme-linked immunosorbent assay (ELISA) kits #IGF31-K01 (Eagle Biosciences, Inc., Amherst, NH, USA) and #SK00033-02 (Aviscera Bioscience Inc., Santa Clara, CA, USA), respectively.

Techniques:

Journal: Blood research

Article Title: Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

doi: 10.5045/br.2020.55.1.35

Figure Lengend Snippet: (A) MTT reduction curve for Hep-2 cells cultured in the presence of 10% FBS, 10% HPL, and 5% HPL and in SFM ( P

Article Snippet: Measurement of growth factors in HPL and FBS samples For HPL and FBS (#S1810-100, Biowest, Riverside, MO, USA) samples, the concentrations of IGF-1 and TGF-β1 were analyzed using enzyme-linked immunosorbent assay (ELISA) kits #IGF31-K01 (Eagle Biosciences, Inc., Amherst, NH, USA) and #SK00033-02 (Aviscera Bioscience Inc., Santa Clara, CA, USA), respectively.

Techniques: MTT Assay, Cell Culture

Journal: Cytotechnology

Article Title: Catalyser-21TM, a mineral water derived from leaf soil, inhibits tumor cell invasion and angiogenesis

doi: 10.1007/s10616-007-9073-4

Figure Lengend Snippet: Detection of H 2 O 2 scavenging activity of Catalyser-21 TM . Cultured HeLa cells were pretreated for 30 min with MEM containing 10% FBS with 3% Catalyser-21 TM , then incubated with 5 μM DCFH-DA for 30 min at 37°C. The fluorescence intensity of DCF was measured with a flow cytometer. The fluorescence intensity relative to that of control cells is presented as curves. Curve (a) is the fluorescence intensity obtained from control HeLa cells. Curve (b) is the fluorescence intensity obtained from HeLa cells treated with 3% Catalyser-21 TM . H 2 O 2 scavenging activity was judged positive, as the Catalyser-21 TM -treatment curve (b) was shifted to the left compared with the control curve (a)

Article Snippet: HeLa cells (a human cervical cancer cell line), HT1080 cells (a human fibrosarcoma cell line), and TIG-1 cells (normal human cells) were obtained from Health Science Research Resources Bank and cultured in modified Eagle’s medium (MEM) supplemented with 10% fetal bovine serum (FBS) (Biowest, France).

Techniques: Activity Assay, Cell Culture, Incubation, Fluorescence, Flow Cytometry, Cytometry

Journal: Cytotechnology

Article Title: Catalyser-21TM, a mineral water derived from leaf soil, inhibits tumor cell invasion and angiogenesis

doi: 10.1007/s10616-007-9073-4

Figure Lengend Snippet: Suppression of VEGF transcription ( A ) and protein secretion ( B ) in HeLa cells by Catalyser-21 TM . ( A ) HeLa cells were cultured for 24 h in MEM containing 10% FBS. The culture medium was then removed and serum-free MEM containing 3% Catalyser-21 TM was added, followed by an additional 24 h incubation. VEGF and GAPDH transcripts were detected by RT-PCR using total RNA isolated from the treated and untreated HeLa cells and appropriately designed primers. Lane a, DNA fragments were amplified from total RNA of untreated HeLa cells. Lane b, DNA fragments were amplified from total RNA of 3% Catalyser-21 TM -treated HeLa cells. Amplified products were resolved by agarose gel electrophoresis and photographs were documented by a digital camera. Recorded images were analyzed by the NIH Image analyzer program (Image 1.62f ) using a personal computer. Values below the panel were normalized by arbitrarily setting the density of the VEGF 165 and VEGF 121 bands of untreated HeLa cells to 1.0. GAPDH transcripts were used as an internal control for cellular activity. ( B ) HeLa cells were treated as above and each supernatant was collected to measure VEGF secretion. The procedure for VEGF protein measurement is described in the Materials and methods. Differences were analyzed by Student’s t test (values are the mean ± SD; n = 3) and the asterisk represents a significant difference compared with the control (* p

Article Snippet: HeLa cells (a human cervical cancer cell line), HT1080 cells (a human fibrosarcoma cell line), and TIG-1 cells (normal human cells) were obtained from Health Science Research Resources Bank and cultured in modified Eagle’s medium (MEM) supplemented with 10% fetal bovine serum (FBS) (Biowest, France).

Techniques: Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification, Agarose Gel Electrophoresis, Activity Assay

Journal: Cytotechnology

Article Title: Catalyser-21TM, a mineral water derived from leaf soil, inhibits tumor cell invasion and angiogenesis

doi: 10.1007/s10616-007-9073-4

Figure Lengend Snippet: Effect of Catalyser-21 TM on HeLa cell proliferation. HeLa cells were cultured in a 24-well microtiter plate with MEM containing 10% FBS for 24 h. The culture medium was then removed and replaced with MEM containing 10% FBS and various concentrations (3–50%) of Catalyser-21 TM , and the cells were incubated for another 24 h. A WST-1 assay was performed, and the data are expressed as the mean ± SD of three independent experiments

Article Snippet: HeLa cells (a human cervical cancer cell line), HT1080 cells (a human fibrosarcoma cell line), and TIG-1 cells (normal human cells) were obtained from Health Science Research Resources Bank and cultured in modified Eagle’s medium (MEM) supplemented with 10% fetal bovine serum (FBS) (Biowest, France).

Techniques: Cell Culture, Incubation, WST-1 Assay

Journal: Cytotechnology

Article Title: Catalyser-21TM, a mineral water derived from leaf soil, inhibits tumor cell invasion and angiogenesis

doi: 10.1007/s10616-007-9073-4

Figure Lengend Snippet: Suppression of MMP-2 expression ( A ) and invasiveness ( B ) of HT1080 cells by Catalyser-21 TM . ( A ) HT1080 cells were cultured for 24 h with MEM containing 10% FBS. The culture medium was replaced by fresh serum-free MEM containing Catalyser-21 TM and the cells were incubated for another 24 h. Total RNA was purified from Catalyser-21 TM -treated and untreated HT1080 cells and used for RT-PCR templates. Amplified products were resolved by agarose gel electrophoresis and analyzed as described in the legend of Fig. 3. Lane a, DNA fragments amplified from total RNA of untreated HT1080 cells. Lane b, DNA fragments amplified from total RNA of 3% Catalyser-21 TM -treated HT1080 cells. Values below the panels were normalized by arbitrarily setting the density of the MMP-2 and GAPDH bands of untreated HT1080 cells to 1.0. GAPDH transcripts were used as an internal control for cellular activity. ( B ) HT1080 cells were pretreated with Catalyser-21 TM for 24 h and then inoculated at 1 × 10 5 cells/well of the Matrigel chamber. The chambers were incubated at 37°C for 12 h, after which the cells that had invaded the lower collagen surface were fixed and stained with Diff-Quick (Sysmex, IL, USA). The invading cells were counted in three random fields under a light microscope. Differences were analyzed by Student’s t test (values are the mean ± SD; n = 3) and the asterisk represents a significant difference compared with the control (* p

Article Snippet: HeLa cells (a human cervical cancer cell line), HT1080 cells (a human fibrosarcoma cell line), and TIG-1 cells (normal human cells) were obtained from Health Science Research Resources Bank and cultured in modified Eagle’s medium (MEM) supplemented with 10% fetal bovine serum (FBS) (Biowest, France).

Techniques: Expressing, Cell Culture, Incubation, Purification, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Activity Assay, Staining, Diff-Quik, Light Microscopy

Journal: Cancers

Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

doi: 10.3390/cancers12082085

Figure Lengend Snippet: In vitro lactic acidosis affects IFN-α production by pDCs. pDCs purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 plus lactic acid (10 mM; 15 mM; 20 mM) ( n = 7; ( A )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5) ( n = 7; ( B )) for 24 h. pDCs were stimulated with R848 for 2 h. Intracellular IFN-α was analyzed by flow cytometry. Aligned dot plot graphs show the percentages of IFN-α + pDCs evaluated on BDCA-2 + /CD123 + cells. Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p

Article Snippet: Isolated pDCs and T lymphocytes (5 × 105 cells/mL) were cultured in RPMI 1640 medium (Biochrom GmbH, Holliston, MA, USA) with 10% FBS (Biochrom GmbH, Holliston, MA, USA), and 20 ng/mL human IL-3 (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to pDCs’ culture medium. pDCs and T lymphocytes were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS (Biochrom GmbH, Holliston, MA, USA), containing lactic acid (LA) (Sigma-Aldrich, St. Louis, MO, USA) or sodium lactate (NaL) (Alfa Aesar, Haverhill, MA, USA) at the concentrations of 10 mM, 15 mM, and 20 mM.

Techniques: In Vitro, Purification, Cell Culture, Flow Cytometry

Journal: Cancers

Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

doi: 10.3390/cancers12082085

Figure Lengend Snippet: Lactic acidosis affects the viability of pDCs and T cells. pDCs and T cells purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS plus lactic Acid (10 mM; 15 mM; 20 mM) ( n = 7 ( A – C ); n = 6, ( D – F )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5 ( n = 6 ( G – I ); n = 7, ( J – L )) for 24 h. IL-3 was added to pDCs’ culture. The cellular viability was analyzed by annexin V/SYTOX AADvanced staining in flow cytometry. Aligned dot plot graphs show the percentages of dead ( A , D , G , J ), early apoptotic ( B , E , H , K ), and late apoptotic or necrotic cells ( C , F , I , L ). Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p

Article Snippet: Isolated pDCs and T lymphocytes (5 × 105 cells/mL) were cultured in RPMI 1640 medium (Biochrom GmbH, Holliston, MA, USA) with 10% FBS (Biochrom GmbH, Holliston, MA, USA), and 20 ng/mL human IL-3 (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to pDCs’ culture medium. pDCs and T lymphocytes were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS (Biochrom GmbH, Holliston, MA, USA), containing lactic acid (LA) (Sigma-Aldrich, St. Louis, MO, USA) or sodium lactate (NaL) (Alfa Aesar, Haverhill, MA, USA) at the concentrations of 10 mM, 15 mM, and 20 mM.

Techniques: Purification, Cell Culture, Staining, Flow Cytometry

Journal: Cancers

Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

doi: 10.3390/cancers12082085

Figure Lengend Snippet: Frequency of interferon alpha (IFN-α) and CXCL10-producing plasmacytoid dendritic cells (pDCs) in chemo-naïve MM patients and HD. Representative dot plots of R848-stimulated IFN-α + and CXCL10 + pDC subsets obtained from HD and MM patients are shown ( A ). PBMCs were isolated from peripheral blood of HD ( n = 25) and MM patients ( n = 29). Total PBMCs were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 and stimulated with R848 or IMQ for 2 h ( B , D ) and 6 h ( C , E ), and with CpG-ODN 2216 for 6 h ( B – E ). IFN-α ( B , D ) and CXCL10 ( C , E ) were analyzed by intracellular flow cytometry staining. Scatter dot plot graphs illustrate the percentages of positive pDCs evaluated on BDCA-2 + /CD123 + cells. Subgroup analysis of the MM cohort illustrating the frequency of IFN-α + and CXCL10 + pDCs in M1a-c categories ( D , E ). Median and IQR are shown in ( B , C ). Mean and SD are shown in ( D , E ). The statistical significance was calculated by Wilcoxon–Mann–Whitney test ( B , C ) and by a Student’s t -test ( D , E ). * p

Article Snippet: Isolated pDCs and T lymphocytes (5 × 105 cells/mL) were cultured in RPMI 1640 medium (Biochrom GmbH, Holliston, MA, USA) with 10% FBS (Biochrom GmbH, Holliston, MA, USA), and 20 ng/mL human IL-3 (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to pDCs’ culture medium. pDCs and T lymphocytes were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS (Biochrom GmbH, Holliston, MA, USA), containing lactic acid (LA) (Sigma-Aldrich, St. Louis, MO, USA) or sodium lactate (NaL) (Alfa Aesar, Haverhill, MA, USA) at the concentrations of 10 mM, 15 mM, and 20 mM.

Techniques: Isolation, Cell Culture, Flow Cytometry, Staining, MANN-WHITNEY

Journal: Human Gene Therapy Methods

Article Title: Sodium Chloride Enhances Recombinant Adeno-Associated Virus Production in a Serum-Free Suspension Manufacturing Platform Using the Herpes Simplex Virus System

doi: 10.1089/hgtb.2016.151

Figure Lengend Snippet: Increased rAAV9 production after sodium chloride supplementation for other rAAV serotypes, genes of interest, and/or cell types. Total transducing units (A) and total vector genomes (B) were evaluated for rAAV2-GFP, rAAV5-GFP, and rAAV9-Des-GAA produced by rHSV coinfection in shaker flasks (50-ml working volume) in the presence of increased sodium chloride concentration ( n = 2 for all). (C) Impact of increased sodium chloride on rAAV9-GFP production in BHK cells grown in DMEM with 5% FBS (BHK) or adapted to serum-free medium (BHK-SFX). (D) Total rAAV9-GFP vector genomes produced in BHK and BHK-SFX cells by rHSV coinfection in the absence or presence of optimal salt supplementation (30 and 60 m M , respectively). * p

Article Snippet: Cell lines Suspension BHK21C13-2P (BHK) cells were obtained from Sigma-Aldrich (St. Louis, MO) and maintained in Dulbecco's modified Eagle's medium (DMEM; HyClone/GE Healthcare Life Sciences, Logan, UT) supplemented with 5% FBS (Corning, Corning, NY) and 1% antibiotic/antimycotic (Gibco/Thermo Fisher Scientific, Waltham, MA).

Techniques: Plasmid Preparation, Produced, Concentration Assay

Journal: Human Gene Therapy Methods

Article Title: Sodium Chloride Enhances Recombinant Adeno-Associated Virus Production in a Serum-Free Suspension Manufacturing Platform Using the Herpes Simplex Virus System

doi: 10.1089/hgtb.2016.151

Figure Lengend Snippet: Characterization of rAAV vector made in Expi293F suspension system. (A and B) rAAV9-GFP was produced by rHSV infection in adherent HEK293 cells grown in 10-chamber CellSTACKs (CS10) under optimal conditions (approximately 3 × 10 9 cells) either in DMEM containing 5% FBS alone ( n = 4) or supplemented with 60 m M sodium chloride ( n = 6), or in a 3-liter suspension culture containing 3 × 10 9 Expi293F cells ( n = 5). Total vector genome (A) and transducing unit (B) yields are shown. Total purified vector genomes are represented relative to units produced per cell (C) and per liter of medium (D) . (E) The vector genome-to-transducing unit (VG/TU) ratio was calculated for each sample, and the averaged values for each group are shown. (F) Coomassie staining of rAAV9-GFP final stocks produced in HEK293 CS10 in the absence of salt supplementation (lane 1), or supplemented with sodium chloride (lane 2), or produced in Expi293F 3-liter cultures supplemented with sodium chloride (lanes 3–7). A total of 10 11 VG was loaded per well. * p

Article Snippet: Cell lines Suspension BHK21C13-2P (BHK) cells were obtained from Sigma-Aldrich (St. Louis, MO) and maintained in Dulbecco's modified Eagle's medium (DMEM; HyClone/GE Healthcare Life Sciences, Logan, UT) supplemented with 5% FBS (Corning, Corning, NY) and 1% antibiotic/antimycotic (Gibco/Thermo Fisher Scientific, Waltham, MA).

Techniques: Plasmid Preparation, Produced, Infection, Purification, Staining