cell type: Human malignant choroid plexus papilloma (HIBCPP) cells infected with: none (control) pathology: control
Treatment protocol
Cells were seeded on inverted cell culture inserts and transferred to phenol red free DMEM/F-12 with 4 mM L-Glutamine and 15 mM HEPES supplemented with 5 µg ml-1 insulin and 1% fetal calf serum when TEER values reached 60 Ω * cm2. Infection from the basolateral side (mimicking the blood facing side in vivo) was carried out the next day when TEER values ranged around 500 Ω * cm2. Using the inverted cell culture insert system infection from the upper compartment simulates the pathophysiological situation in vivo, when pathogens invade the CSF from the blood. HIBCPP cells were infected with either strain MC58, MC58siaD- or α14 at a multiplicity of infection (MOI) of 10 at 37°C and 5% CO2 atmosphere for the indicated periods of time with antibiotical killing of the bacteria after 4 h and prolonged infection up to 24 h.
Growth protocol
HIBCPP cells were cultured in DMEM/F-12 (Ham) 1x with 4 mM L-Glutamine and 15 mM HEPES supplemented with 5 µg ml-1 insulin, 100 U ml-1 penicillin and 100 µg ml-1 streptomycin as well as 15% heat inactivated fetal calf serum (FCS). For inverted cell culture insert based experiments 0.7 * 105 cells were seeded on filter inserts (pore diameter 3.0 µm, pore density 2.0 * 106 pores per cm2, growth area 0.33 cm2, from either Millipore, Schwalbach, Germany or Greiner Bio-one, Frickenhausen, Germany) that were flipped over and placed in a medium flooded 12-well plate.
Extracted molecule
total RNA
Extraction protocol
Following exposure to bacteria, cells were washed twice with PBS to exclude bacteria. Total RNA from HIBCPP cells was extracted using the QIAGEN RNeasy® Mini or Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. During RNA purification an on-column DNAse digestion (RNase-Free DNase Set, Qiagen, Hilden, Germany) was performed to avoid DNA carryover. RNA purity was evaluated by spectrophotometer (ND1000, Peqlab Biotechnoloy, Erlangen, Germany) and RNA quality of the microarray samples was additionally assessed using the Agilent RNA 6000 Nano Kit according to the manufacturer’s instructions and the Agilent Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany). Samples with RNA integrity numbers (RIN) higher than 9.8 were used for microarray analysis.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol.
Hybridization protocol
Hybridization (16h x 45°C) was processed according to the standard Affymetrix protocol.
Scan protocol
Affymetrix GeneArray Scanner3000
Description
uninfected HIBCPP control cells; replicate 3 HIBCPP-untreated #3
Data processing
The data were analyzed with a commercial software called JMP Genomics, version 4, from SAS. Gene expression profiling was performed using arrays of HG-U133_Plus_2 -type from Affymetrix. A Custom CDF Version 13 with unigene based gene definitions was used to annotate the arrays. The Raw fluorescence intensity values were normalized applying quantile normalization.
