fbs  (Atlanta Biologicals)

Journal: PLoS ONE

Article Title: Mechanism of Fibronectin Binding to Human Trabecular Meshwork Exosomes and Its Modulation by Dexamethasone

doi: 10.1371/journal.pone.0165326

Figure Lengend Snippet: Trabecular meshwork (TM) exosomes bind fibronectin on the external surface. Serum-free medium was conditioned for 2.5 hours with primary TM cell cultures. Conditioned media was split in half and either exosome-depleted FBS (10% final v:v) or purified human plasma fibronectin (Fn, 2.5μg/ml [final]) was added to one portion. Exosomes were prepared from both portions (and from unconditioned media + FBS or purified Fn) and Fn content was assessed by Western blot. The blots shown represent the results from a minimum 3 biological replicates (cell strains isolated from different human donors).

Article Snippet: In some experiments, purified human plasma fibronectin (#F0895, Sigma-Aldrich) or exosome-depleted FBS (#S11550, Atlanta Biologicals, Lawrenceville, GA; FBS spun overnight at 100,000 g ) was added to cleared supernatant for a final concentration of 2.5 μg/ml or 10% v:v, respectively.

Techniques: Purification, Western Blot, Isolation

Journal: Antioxidants & Redox Signaling

Article Title: Disruption of Pyridine Nucleotide Redox Status During Oxidative Challenge at Normal and Low-Glucose States: Implications for Cellular Adenosine Triphosphate, Mitochondrial Respiratory Activity, and Reducing Capacity in Colon Epithelial Cells

doi: 10.1089/ars.2010.3489

Figure Lengend Snippet: MQ-induced changes in cellular ATP and cytosolic lactate. HT29 cells (1 × 10 4 ) were seeded in 96-well plates and grown overnight at 37°C. The next day, the culture media were replaced by fresh phenol-red and fetal bovine serum-free Dulbecco's modified essential medium containing 1.2, 4.6, or 18.3 m M glucose, and the cells were exposed to 50 or 200 μ M MQ. (A) In time course experiments, cells were treated with MQ for 0–60 min. ATP was measured with a luciferin/luciferase bioluminescence Promega kit according to manufacturer's protocol. Each experiment was conducted in quadruplicates and ATP contents are expressed as percent relative to untreated controls (100%). Results are mean ± SEM for six cell preparations. (B) The contribution of mitochondria-derived ( upper panel ) and glycolysis-derived ( bottom panel ) ATP to total ATP levels was determined in cells treated with antimycin A (2 μ M ) or 2-deoxyglucose (2DG), respectively. Concentrations of 2DG, namely, 3, 11.5, and 45.8 m M were 2.5 × higher than the respective glucose concentrations. Results are mean ± SEM for n = 6 for 18.3 m M glucose, and n = 4 each for 4.6 and 1.2 m M glucose. * p

Article Snippet: Antibiotic/antimycotic, trypsin, L-glutamine, McCoys' medium, and Dulbecco's modified essential medium (DMEM) were from Gibco Corporation, and fetal bovine serum (FBS) was from Atlanta Biologicals.

Techniques: Modification, Luciferase, Derivative Assay

Journal: Journal of Cancer

Article Title: Ex Vivo Derived Primary Melanoma Cells: Implications for Immunotherapeutic Vaccines

doi: 10.7150/jca.6625

Figure Lengend Snippet: Melanoma cells display invasive properties in vitro. Melanoma cells, 2.5 X 10 4 cells, were resuspended in RPMI (500µl) containing 1% FBS and allowed to invade for 18 hours. All of the primary melanoma cells (MEL-2, MEL-V, 3MM, KFM, and GLM-2) displayed differential invasive properties as did the established melanoma cell (SK-Mel-28, SK-Mel-37, and SK-Mel-103). The data is represented as invasion index, which is calculated as the percent of cells invading through the growth factor reduced matrigel invasion chambers relative to the cells migrating through control membrane. Each experiment was repeated three times with similar results observed and the standard deviation shown above each sample represents three replicates in one experiment.

Article Snippet: SK-Mel-28, SK-Mel-37, and SK-Mel-103 are established human melanoma cell line, which were generously gifted to us by Jedd D. Wolchok, MD, PhD (Memorial Sloan-Kettering Cancer Center, New york, NY) and were cultured in RPMI supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA), 2mM L-glutamine (Mediatech), penicillin 10,000IU/ml, streptomycin 10,000ug/ml, and Normocin™ 100μg/ml (Invivogen).

Techniques: In Vitro, Standard Deviation

Journal: Viruses

Article Title: Tet-Inducible Production of Infectious Zika Virus from the Full-Length cDNA Clones of African- and Asian-Lineage Strains

doi: 10.3390/v10120700

Figure Lengend Snippet: Comparison of cDNA-derived MR766 and FSS13025 virus growth. Vero cells (2.5 × 10 5 /well) in 12-well plates were co-transfected with 1 µg pTight-ZIKV/MR766 or pTight-ZIKV/FSS13025 DNA and 1 µg pTet-On vector, followed by the addition of 1 µg/mL Doxycycline to cell culture media. Infectious virus titers in the supernatants collected at day 3 (MR766) or day 6 (FSS13025) were quantified by a plaque assay. ( A ) Plaque formation by cDNA-derived ZIKV/MR766 and ZIKV/FSS13025 viruses. Vero cells in 12-well cell culture plate were infected with 30 PFU of either MR766 or FSS13025. Plaques were stained and visualized after 4 days (for MR766) or 6 days (for FSS13025) p.i. ( B ) Growth curves of cDNA-derived MR766 and FSS13025 viruses. To compare the growth ability between MR766 and FSS13025, 4 × 10 5 Vero cells seeded in 6-well plates were infected with MR766 or FSS13025 virus at 0.01 MOI at 37 °C for 1 h. Upon washing with phosphate-buffered saline (PBS) three times, infected Vero cells were incubated with 2 mL DMEM containing 10% FBS. At day 1, 2, and 3 post-infection, virus in the supernatant was collected and stored at −80 °C. Virus yields at different time points were determined by a plaque assay. Data points represent the mean titer of triplicates. Statistical significance was analyzed by Student’s t -test: ** p

Article Snippet: Vero and C6/36 (CRL-1660) cell lines were obtained from America type culture collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Atlanta, GA, USA), 0.1 mM nonessential amino acids, penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA and HyClone, Logan, UT, USA) at 37 °C in a 5% CO2 incubator.

Techniques: Derivative Assay, Transfection, Plasmid Preparation, Cell Culture, Plaque Assay, Infection, Staining, Incubation