After isolation, NK cells were cultured for the indicated time points in RPMI 1640 (Lonza Verviers, Belgium) supplemented with 10% Fetal Bovine Serum (FBS, Voden Medical S.p.a. Meda MB, Italy), antibiotic mixture (0.05 mg/mL penicillin, 0.05 mg/mL streptomycin Lonza, Verviers, Belgium), and 100 U/mL recombinant human IL-2 (Proleukin, Novartis Basilea, Switzerland) at 2X 106cells/mL in round bottom 96-well microtiter plates. The cultures were performed either under normoxic conditions in a humidified incubator containing 20% O2, 5% CO2, and 75% N2 or under hypoxic conditions. Hypoxic conditions were obtained by culturing cells in a sealed anaerobic workstation incubator (Ruskinn, INVIVO2 400, CARLI Biotec, Roma, Italy), incorporating a gas mixing system (Ruskinn Gas Mixer Q) and flushed with a mixture of 1% O2, 5% CO2, and 94% N2.
Extracted molecule
total RNA
Extraction protocol
Total RNA was purified from different donor-derived NK cells using the RNeasy MiniKit from Qiagen (Milano, Italy). RNA was controlled for integrity by nanoelectrophoresis with an Agilent 2100 Bioanalyzer (Agilent Technologies Europe, Waldbroon, Germany), quantified by spectrophotometry using a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, USA).
Label
biotin
Label protocol
The RNA was reverse-transcribed into double-stranded cDNA on a GeneAmp PCR System 2700 thermal cycler (Applied Biosystems, Milano) using the one-cycle cDNA synthesis kit (Affymetrix, Milano). cDNA derived from three donors/time point was purified and biotin labeled using the GeneChip IVT kit (Affymetrix). Labeled cRNA was fragmented according to Affymetrix’s instructions.
Hybridization protocol
Fragmented cRNA was hybridized on the Affymetrix HG-U133 plus 2.0 GeneChips (Genopolis Corporation, Milano) containing 54,000 probe sets (coding for 47,000 transcripts and variants, including 38,500 unique human genes) on a single array. Chips were stained with streptavidin-phycoerythrin (Invitrogen Life Technologies, Milano) as described.
Scan protocol
GeneChips were scanned using an Affymetrix GeneChip Scanner 3000.
Description
NK cells from healthy donors were isolated from PB mononuclear cells using RosetteSep NK Cell Enrichment Cocktail (StemCell Technologies, 15025 Vancouver, Canada), according to a protocol approved by the Regional Ethics Committee for Human Experimentation, Written informed consent was obtained from all the subjects enrolled in the study in adherence with the Declaration of Helsinki. Only preparations displaying more than 95% of CD56+ CD3- CD14- NK cells were selected for the experiments. After isolation, NK cells were cultured for the indicated time points in RPMI 1640 (Lonza Verviers, Belgium) supplemented with 10% Fetal Bovine Serum (FBS, Voden Medical S.p.a. Meda MB, Italy), antibiotic mixture (0.05 mg/mL penicillin, 0.05 mg/mL streptomycin Lonza, Verviers, Belgium), and 100 U/mL recombinant human IL-2 (Proleukin, Novartis Basilea, Switzerland) at 2X 106cells/mL in round bottom 96-well microtiter plates. The cultures were performed either under normoxic conditions in a humidified incubator containing 20% O2, 5% CO2, and 75% N2 or under hypoxic conditions. Hypoxic conditions were obtained by culturing cells in a sealed anaerobic workstation incubator (Ruskinn, INVIVO2 400, CARLI Biotec, Roma, Italy), incorporating a gas mixing system (Ruskinn Gas Mixer Q) and flushed with a mixture of 1% O2, 5% CO2, and 94% N2.
Data processing
Data were processed by RMA normalization utilizing the ‘Affy’ R package. Probesets with an Absent call in all samples were filtered out. Statistical analysis using paired t test was performed to identify differentially expressed genes. We corrected the p value for multiple hypothesis testing by Benjamini-Hochberg method to false discovery rate control. Only gene differences that passed the test at a confidence level of 95% (P<0.05) and a false discovery rate of 0.05% were considered significant. Fold-change (FC) was calculated as the ratio between the average expression level under hypoxia and normoxia. Genes were defined as being differentially regulated by hypoxia if they exhibited more than 2-fold increase in gene expression or down-regulated if they showed less than 0.5-fold change compared with normoxic cultures. We converted the Affymetrix probe sets into the corresponding gene symbol by Netaffx tool. When multiple probe sets were associated with the same gene symbol, the probe set with the highest expression signal was considered. Biological processes were assessed by DAVID Gene Ontology (GO) enrichment analysis (http://david.niaid.nih.gov). The significant GO terms were defined as p value <0.05 and FDR <0.05.
