Computational protocol: A tryptophan derivative, ITE, enhances liver cell metabolic functions in vitro

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Protocol publication

[…] Alginate encapsulation was performed as previously described () with modifications. Briefly, the Huh7 and C3A cells were suspended in 2.0% sodium alginate solution (154 mM NaCl, 10 mM HEPES, pH 7.4). Each mixture was sprayed at a flow rate of 9.5 ml/min through a 300 μm nozzle using an electrostatic microencapsulator unit (Nisco Engineering AG, Zurich, Switzerland). The vibration frequency of the nozzle was kept at 0.30 kHz. The alginate droplets were collected in a calcium chloride gelation bath (154 mM NaCl, 10 mM HEPES, 115 mM CaCl2, pH 7.4), followed by 10 min of gelling and normal saline washes for three times. Eventually, the Huh7 and the C3A microspheres with the designed 800 μm diameter were produced. [...] Briefly, total RNA was extracted from the C3A cells treated with 0.2 μM ITE using TRIzol reagent (Cat. no. 15596-108; Life Technologies, Carlsbad, CA, USA) following the manufacturer's instructions, and the RIN was tested to inspect RNA integrity using an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc., Santa Clara, CA, USA). Qualified total RNA was further purified using the RNeasy micro kit (Cat. no. 74004) and the RNase-Free DNase Set (Cat. no. 79254) (both from Qiagen). Total RNA was amplified, labeled and purified using the GeneChip 3′ IVT PLUS Reagent kit to obtain biotin-labeled cRNAs (Cat. no. 902416; Affymetrix, Inc., Santa Clara, CA, USA) following the manufacturer's instructions. Array hybridization and washes were performed using the GeneChip® Hybridization, Wash and Stain kit (Cat. no. 900720) in a hybridization oven 645 (Cat. no. 00-0331-220V) and Fluidics Station 450 (Cat. no. 00-0079) (all from Affymetrix, Inc.) according to the manufacturer's instructions.Gene expression profiling was conducted by Shanghai Biotechnology Corporation using the Affymetrix PrimeView™ Human Gene Expression Array (Affymetrix, Inc.). All data were analyzed according to the manufacturer's instructions. Raw data originated from Affymetrix CEL files were normalized by RMA background correction and values were log2 transformed. Comparison of the data sets by the t-test showed that 1,472 of the total of 49,293 probe-sets (2.99%) were differentially expressed (|fold changes| ≥2). In order to analyze the enrichment of P-values of each GO term, we used Fisher's exact test to calculate P-values and R package stats to calculate FDR (q-value) by the BH method (www.r-project.org). […]

Pipeline specifications

Software tools Nozzle, PrimeView
Applications Miscellaneous, Gene expression microarray analysis
Diseases Carcinoma, Hepatocellular, Liver Diseases
Chemicals Tryptophan, Urea