Sex: male subjectid: OFP cell line: Caco-2 cell line origin: Caucasian colon adenocarcinoma passage nr: 30-60 days of differentiation on transwells: 21 treatment: incubated with Frutalose OFP (OFP) for 6 hours
Treatment protocol
Glycerol stocks of Lactobacillus acidophilus W37 (W37), Lactobacillus brevis W63 (W63), and Lactobacillus casei W56 (W56) (Winclove, Amsterdam, The Netherlands) were produced from bacteria grown anaerobically overnight in MRS medium. Upon use, glycerol stocks were washed with PBS and resuspended in cells culture medium, brought to 37°C, to reach 10^7 CFU/mL. Inulin-type fructans and oligofructose were solubilized at 0.5 mg/mL in medium and filtered (0.2 m) to eliminate possible contaminations. The cell medium was replaced the day before the exposure experiments. Medium was removed from the apical and basal compartments and diluted samples were added in to the apical compartment while fresh DMEM+10%FBS medium was added to the basal compartment. In each experiment, the compound- and control samples were exposed to Caco-2 cells in triplicate and two or three independent exposure experiments were performed (= independent biological replicates). After 6h incubation, the Caco-2 cells were lysed with 300 μL TRIzol and the triplicates in each experiment were pooled for RNA isolation.
Growth protocol
ATCC derived Caco-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-Invitrogen, Bleiswijk, The Netherlands) with 4.5 g/L glucose, 0.58 g/L glutamine, no pyruvate, supplemented with 10% heat inactivated FBS (Hyclone Perbio, Etten-Leur, The Netherlands) and used with passage numbers between 30 and 60. For transwell assays, 0.33x105 cells were grown on ThinCert transwells with 33.6 mm2 membranes and 0.4 μm pores in 24-well suspension culture plates. Cells were grown for 21 days at 5% CO2 and 37°C and apical (150 μl) and basolateral (700 μl) medium were replaced three times per week.
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared from the Caco-2 cells using TRIzol reagent, whereafter purified total RNA was isolated using Qiagen RNEasy columns. RNA integrity was checked on chip analysis (Agilent 2100 bioanalyzer, Agilent Technologies, Amsterdam, the Netherlands) according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no chromosomal peaks or RNA degradation products (RNA Integrity Number > 8.0).
Label
biotin
Label protocol
One hundred nanogram of RNA was used for whole transcript cDNA synthesis with the Ambion WT expression kit [catalog number 4411974] (Applied Biosystems/Life Technologies, Nieuwekerk a/d IJssel, The Netherlands).
Hybridization protocol
Hybridization and washing of the Affymetrix GeneChip Human Gene 1.1 ST peg arrays were performed on a GeneTitan Instrument (Affymetrix, Santa Clara, CA) according to the manufacturer's recommendations.
Scan protocol
Arrays were scanned on an Affymetrix GeneTitan instrument (Affymetrix, Santa Clara, CA).
Description
21 days differentiated Caco-2 cells incubated with Frutalose OFP (OFP) for 6 hours, biological replicate 3
Data processing
Expression estimates were calculated applying the RMA algorithm using the Affymetrix® Expression Console™ Software.
