genotype: SCS-BTA18-q treatment: Escherichia coli time point: 24 hours sample type: heifer with unfavorable QTL allele
Treatment protocol
Heat inactivated S. aureus M60 and E. coli isolates deriving from a milk sample of an udder with bovine mastitis were used for inoculation. Epithelial cells were thawed and cultured under cultivation conditions (37°C, 5% CO2, and 90% humidity) in DMEM/F12 medium containing 100 μg/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml gentamicin, 5 μg/ml amphotericin, 10% FBS and 10 μl/ml ITS for 2 further passages. For pathogen challenge they were seeded in 3 six-well tissue culture plates (Greiner bio-one, Frickenhausen, Germany), one plate for each time point (1, 6 and 24 hours), in a concentration of 300,000 cells/well. Two wells in each plate were prepared for control and two each for S. aureus and E. coli treatment. The medium was replaced by DMEM/F12 supplemented with ITS on the following day. At a confluence of about 70% on the second day after seeding, the medium was refreshed. 100 μl of bacterial-solution representing a multiplicity of infection of 10, was added. 100 μl PBS were used as control treatment of the uninoculated control cells.
Growth protocol
On day 42 of their first lactation all selected heifers were slaughtered. Two samples of about 1.5 x 1.5 x 1.5 cm were taken aseptically from the parenchyma immediately after the animal was slaughtered and were transferred in 200 ml of room-temperate Hank’s balanced salt solution (HBSS; Sigma-Aldrich, Munich, Germany) containing 200 μg/ml penicillin, 200μg/ml streptomycin, 200μg/ml gentamicin and 10μg/ml amphotericin B (Sigma-Aldrich, Munich, Germany). After the tissue was minced and blood as well as milk residues were flushed away with HBSS, the samples were transferred to a digestion mix consisting of 200ml HBSS, 0.5mg/ml collagenase IA, 0.4 mg/ml DNAse type I, 0.5 mg/ml hyaluronidase (enzymes from Sigma-Aldrich, Munich, Germany) and the same antibiotics mentioned above. The cells were placed in a shaking incubator at 37°C for 165 minutes and were separated from connective tissue and non-epithelial cell conglomerates by filtration and centrifugation (500 U; 4min). This procedure was repeated two times each with a decreased pore size of the strainer (smallest one: 100μm, BD Biosciences, Erembodegem, Belgium). After washing and separation, the cells were resuspended in Dulbecco’s modified eagle’s medium nutrient mixture F-12 Ham (DMEM/F12, Sigma-Aldrich, Munich, Germany) containing penicillin, streptomycin, gentamicin, and amphotericin B in the same concentrations as described above, as well as 10% FBS and 10 μl/ml ITS (0.5 mg/ml bovine Insulin, 0.5 mg/ml apo-transferrin, 0.5μg/ml sodium selenite; Sigma-Aldrich, Munich, Germany). The cells were incubated in a 75 cm² tissue culture flask (Greiner bio-one, Frickenhausen, Germany) for 40 min at 37°C, 5% CO2, and 90% humidity. Thereafter the fibroblasts had attached and epithelial cells could be isolated by decanting. Epithelial cells were seeded in 25 cm² tissue culture flasks (Greiner bio-one, Frickenhausen, Germany) and cultured until they reached about 80% confluence. Cells were cryopreserved at -80°C in 1 ml freezing medium containing DMEM/F12, 20%FBS, and 10% DMSO. An immunocytochemical staining was conducted to verify the epithelial origin of the cells. Approximately 90 to 95% off the cells were represented by epithelial cells.
Extracted molecule
total RNA
Extraction protocol
Cells were harvested after 1, 6 and 24 hours and total RNA was extracted with the TriFast reagent as indicated in the manufacturer’s instructions (PEQLAB Biotechnology GmbH, Erlangen, Germany)
Label
biotin
Label protocol
2µg of total RNA were used for preparation of antisense biotinylated RNA using the GeneChip 3’IVT Express kit (Affymetrix, St. Clara, USA)
Hybridization protocol
Following fragmentation, 10 ug of biotinylated cRNA were hybridized for 16 hours at 42°C on Affymetrix GeneChip Bovine Genome Arrays (Affymetrix, St. Clara, USA). GeneChips were washed and stained in the Affymetrix Fluidics Station 400
Scan protocol
GeneChips were scanned using the GeneChip scanner 3000 (Affymetrix, St. Clara, USA)
Description
Gene expression data from E. coli inoculated cells
Data processing
Data were analysed with Affymetrix Expression Console 1.1 software using global scaling to a target signal of 500
Comparative Expression Profiling of E. coli and S. aureus inoculated primary Mammary Gland Cells sampled from Cows with different genetic Predisposition for Somatic Cell Score
