|
| Status |
Public on Mar 22, 2017 |
| Title |
UCN34 5h a2 M1 - 159 A3 |
| Sample type |
RNA |
| |
|
| Source name |
S. gallolyticus UCN34; 5h after phagocytosis by THP-1 macrophages; -60min
|
| Organism |
Streptococcus gallolyticus subsp. gallolyticus |
| Characteristics |
strain: UCN 34
interaction status: after maturation of phagosomes (5h)
time point: 5h
|
| Treatment protocol |
This phagocytosis assay based on the assays of Boleji et al. and Kaneko et al. (13, 32). For Phagocytosis assay 1.5 ´ 106 THP-1 monocytes per well were disseminated in 12-well plates with 50 ng/ml PMA-supplemented medium to differentiate the monocytes into macrophages within three days. On the third day the medium was changed into PMA-free medium after washing the cells twice with Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Scientific, Waltham, USA). An overnight culture of S. gallolyticus subsp. gallolyticus was serial diluted (103 dilution) in RPMI 1640 supplemented with 10 % FCS without AB/AM and the final bacterial titer of the inoculum was determined by plating assay. After washing macrophages with DPBS thrice, the S. gallolyticus subsp. gallolyticus dilution was added and plates were centrifuged 5 min with 400 ´ g to assure attachment of bacteria to macrophages (adhesion to macrophages; -60 min). Phagocytic uptake of bacteria was ensured for 30 min at 37 °C and 5 % CO2 and macrophages were washed thrice with DPBS. RPMI 1640 including 10 % FCS, 1 ´ AB/AM and 200 µg/ml gentamycin was added for 20 min to kill residual extracellular bacteria. Initially after the step (0 h) and after 5 h cells were prepared for RNA extraction.
|
| Growth protocol |
THP-1 cells were cultivated in RPMI 1640 medium (RPMI 1640; Thermo Scientific, Waltham, USA) supplemented with 10 % fetal calf serum (FCS, Pan Biotech; Aidenbach, Germany) and 1 ´ antibiotic/antimycotic solution (AB/AM, Pan Biotech) at 37 °C and 5 % CO2. S. gallolyticus subsp. gallolyticus strains (table 1) were grown overnight in brain heart infusion broth (BHI; Thermo Scientific, Waltham, USA) at 37 °C and 220 rpm. Overnight cultures were used by phagocytosis assays.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Gene expression was analyzed from the two S. gallolyticus subsp. gallolyticus isolates BAA-2069 and UCN 34. RNA from these strains was isolated at three different time points. The first time point was directly after centrifugation (400 × g, 5 min) of the well plates (-60 min). The second RNA extraction was at the timepoint 0 h and the third at the timepoint 5 h. RNA was extracted with the peqGOLD Bacterial RNA Kit (VWR, Radnor, USA). Bacterial cells were resuspended in TE buffer and lysis buffer T and transferred in Lysing Matrix B tubes (MP Biomedicals, Santa Ana, USA). Bacterial cells were disrupted by using Vortex-Genie 2 (scientific industries, New York, USA) for 3 min at full speed. Further RNA extraction was done following the manufacturer’s instructions. RNA was eluted with 30 µl RNase-free water and quantified using the NanoDrop 2000.
|
| Label |
Cy3
|
| Label protocol |
The cDNA and cRNA synthesis including Cy3-labeling, and the microarray hybridization was carried out with the Quick Amp WT Labeling Kit, one-color (Agilent, Santa Clara, USA) according to the manufacturer’s recommendations but with following exceptions. Due to mixed RNA (prokaryotic and eukaryotic) more RNA (75 ng) was used for labeling and 900 ng labeled cRNA were used for hybridization.
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| |
|
| Hybridization protocol |
The Hybridization was also carried out with the Quick Amp WT Labeling Kit, one-color (Agilent, Santa Clara, USA) according to the manufacturer’s recommendations. To achieve sufficient fluorescent signals, hybridization was extended to 38 h. The slides were washed and hybridization was stabilized with Stabilization & Drying solution (Agilent, Santa Clara, USA).
|
| Scan protocol |
After drying, the hybridized microarrays were scanned with the high resolution Agilent microarray scanner G2565CA with a resolution of 5 µm and analyzed with the Feature extraction software (Agilent, Santa Clara, USA).
|
| Description |
long time after phagocytosis
|
| Data processing |
Raw data were quantile-normalized and gene expression data were generated by the Direct Array software (OakLabs, Hennigsdorf, Germany). All log2 values between -1 and 1 were ignored and only statistically significant values (p<0.05) have been taken.
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| |
|
| Submission date |
Mar 21, 2017 |
| Last update date |
Mar 22, 2017 |
| Contact name |
Imke Grimm |
| E-mail(s) |
[email protected]
|
| Organization name |
Herz- und Diabeteszentrum NRW; Universitätsklinikum der Ruhr-Universität Bochum
|
| Department |
Institut für Laboratoriums- und Transfusionsmedizin
|
| Street address |
Georgstraße 11
|
| City |
Bad Oeynhausen |
| ZIP/Postal code |
32545 |
| Country |
Germany |
| |
|
| Platform ID |
GPL23196 |
| Series (2) |
| GSE96862 |
Transcriptome analysis of Streptococcus gallolyticus subsp. gallolyticus in interaction with THP-1 macrophages [UCN 34 5h] |
| GSE96865 |
Transcriptome analysis of Streptococcus gallolyticus subsp. gallolyticus in interaction with THP-1 macrophages |
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