tissue: peripheral blood gender: male age: 1-y-old time: 4 h treatment: 200 µg/mL of GSNO-loaded ENP cell line: THP-1
Growth protocol
THP-1 human monocyte cell line is derived from a blood sample of a one-year old child suffering from acute monocytic leukemia and was obtained from American Type Culture Collection (ATCC, TIB-202TM, Manassas, VA, USA). Cells were grown in RPMI 1640 medium (GIBCO, Invitrogen, Cergy Pontoise, France) supplemented with 10 % of heat-inactivated fetal bovine serum (Eurobio, Les Ullis, France), as well as 100 U/mL of penicillin, 100 µg/mL of streptomycin and 0.25 µg/mL of amphotericin B (all from Sigma, Saint Quentin Fallavier, France). Cells were incubated at 37°C to an atmosphere composed of 95 % air, 5 % CO2 and diluted by half every 3 days.
Extracted molecule
total RNA
Extraction protocol
The quality of the RNA extracted by TRIzol® Reagent (Invitrogen, La Jolla, CA) was determined by spectrophotometry (A260nm/A280nm > 1.8, BioSpec-nano, Shimadzu) and by capillary electrophoresis using RNA 6000 Nano® (RIN > 9, Agilent 2100 Bioanalyser, Santa Clara, CA, USA).
Label
CTP-Cy3
Label protocol
CTP-cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng RNA using the One-Color Microarray-Based Gene Expression Analysis (Agilent, version 6.6) according to manufacturer's instructions, followed by chromatographic purification (RNeasy Mini kit, QIAGEN). Dye incorporation and cRNA yield and quality were checked by spectrophotometry (BioSpec-nano, Shimadzu) and by electrophoresis (Agilent 2100 Bioanalyser).
Hybridization protocol
Six hundred ng of CTP-Cy3-labelled cRNA (specific activity > 10 pmol Cy3 / µg cRNA) was fragmented at 60°C for 30 min in a reaction volume of 25 µL containing 25 x Agilent fragmentation buffer and 10 x Agilent blocking agent following the manufacturer’s instructions. On cRNA fragmentation completion, 25 µL of 2 x Agilent hybridization buffer were added to the fragmentation mixture and cRNA were hybridized to SurePrint G3 Human GE v2 8x60K (Agilent) for 17 h at 65°C in a rotating hybridization oven. After hybridization, microarrays were washed 1 min at room temperature with GE Wash Buffer 1TM and 1 min with 37°C GE Wash buffer 2TM (Agilent), and immediately dried by brief immersion in acetonitrile.
Scan protocol
Slides were scanned immediately after washing using Agilent DNA Microarray Scanner (G2505C) by setting: (i) one color scan channel for 8x60k array slides, (ii) scan area of 61x21.6 mm, (iii) scan resolution of 3 µm, (iv) dye channel to Green, (v) Tiff file dynamic range of 20 bits and (vi) Green PMT to 100 %.
Description
Gene expression in THP-1 cells after 4 h in presence of GSNO-loaded ENP exposure (200 µg/mL)
Data processing
The scanned images were analyzed by Feature Extraction version 11.0.1.1 (Agilent) using default parameters (protocol GE1_1100_Jul11 and Grid: 039494_D_F_20120628) to subtract background and to obtain spatially detrended processed signal intensities. Features flagged in Feature Extraction as Non-uniform outliers Feature were excluded.
Transcriptome study of THP-1 human monocytes following exposure for 4 h or 24 h to 50 uM S-Nitrosoglutathione, 50 and 200 ug/ml S-Nitrosoglutathione-loaded polymeric and empty Eudragit RL nanoparticles
