Mouse strain: vitamin D3 receptor mutant (mut) and wild-type (wt) (see Erben, R. G., Soegiarto, D. W., Weber, K., Zeitz, U., Lieberherr, M., Gniadecki, R., Moller, G., Adamski, J. and Balling, R., Deletion of deoxyribonucleic acid binding domain of the vitamin D receptor abrogates genomic and nongenomic functions of vitamin D. Mol Endocrinol 2002. 16: 1524-1537. Gender: male (mut and wt); Age: 9 weeks (mut and wt); Source tissue: bone marrow; Cell type: in vitro generated dendritic cells.
Biomaterial provider
Kindly provided by A. Lengeling, German Research Centre for Biotechnology, GBF, Braunschweig, Germany.
Treatment protocol
Dendritic cells (DC) from VDRmut and control VDRwt mice were generated as described in the Growth Protocol section. On day 7 differentiation DC were pre-treated for 30 min with 1a,25-dihydroxyvitamin D3 (VD3) at 10^-6M, or left untreated. TNFa (50 ng/ml) was added where appropriate, and cells incubated further for 4 h.
Growth protocol
Dendritic cells (DC) were generated as previously described (Hieronymus, T., T. C. Gust, R. D. Kirsch, T. Jorgas, G. Blendinger, M. Goncharenko, K. Supplitt, S. Rose-John, A. M. Muller, and M. Zenke. 2005. Progressive and controlled development of mouse dendritic cells from Flt3+CD11b+ progenitors in vitro. J Immunol 174:2552-2562). Bone marrow was prepared from hind legs of a 9-weeks old (male) VDRmut and VDRwt control mice. Briefly, homogenised cell suspension was diluted to 2x10^6 cells/ml in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and streptomycin sulphate, 50 uM b-mercaptoethanol. Medium was supplemented with the following cytokines/growth factors: 20 U/ml murine SCF, 5 ng/ml hyperIL-6, 25 ng/ml Flt-3L, 40ng/ml human IGF-1 long range, 10^-6 uM dexamethasone and 25 U/ml murine GM-CSF. Cells were then cultured at 37°C in 5% CO2. After 3 days in culture cells were applied to density gradient centrifugation using Ficoll-Hypaque (1,077g/ml) to remove dead cells and debris and to recover factor-dependent progenitor cells. Cells were washed once in complete RPMI medium and adjusted to 2x10^6 cells/ml; above listed growth factors were added again. Medium was renewed every second day and cells were adjusted to 2x10^6 cells/ml, supplementing with growth factors over again. (Cells were maintained in these conditions for 7 days.) At day 7 of culture differentiation of DC from the proliferating progenitor cells was initiated. Cells were washed once in RPMI medium and adjusted to 2x10^6 cells/ml. Cells were seeded in new dishes and murine GM-CSF was added at final concentration of 250U/ml. Medium was renewed every second day, supplementing with 250 U/ml GM-CSF. Since cells typically carry on proliferating in the first 3-4 days of differentiation, they were adjusted to the concentration of 2x10^6 cells/ml until onset of proliferation during every medium change. After 7 days of differentiation from progenitor cells a homogenous population of DC (in suspension; beside strongly adherent macrophages) developed. Non-adherent DC were used for the experimental treatment.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated with RNeasy Midi Kit including DNase digestion (Qiagen).
Label
Phycoerythrin (PE)
Label protocol
5 ug of total RNA was used to generate cRNA using a modified protocol from the Expression Analysis Technical Manual (Affymetrix). RNA was concentrated to >0.5 ug/ul by acid-ethanol precipitation. For the first-strand cDNA synthesis a T7-oligo(dT) primer was firstly hybridised to 8 ug of total RNA by denaturation for 10 min at 70°C, then quick-chilling on ice, followed by incubation for 2 min at 42°C. The reverse transcription reaction was performed using 200U SuperScript RT (Invitrogen) per sample for 1 h at 42°C. The second-strand cDNA synthesis reaction was carried out by firstly incubating the single-stranded cDNA with 10U of DNA ligase, 40U of DNA Polimerase I and 2U of RNase H for 2 h at 16°C, and then additionally for 5 min at 16°C with T4 DNA Polymerase in order to enhance the efficiency of the second-strand synthesis. cDNA was cleaned-up using the QIAquick Spin PCR Purification Kit (Qiagen). Buffer PB was used for binding: this allows removal of fragments smaller than 100 bp, i.e. T7-oligo(dT) primers. DNA was eluted with elution buffer (EB). Double-stranded cDNA was then concentrated again by acid-ethanol precipitation. Synthesis of biotin-labelled cRNA was conducted using T7 Mega Script Kit (Ambion). cDNA template was incubated with dNTPs in the presence of Bio-11-CTP and Bio-11-UTP (Perkin Elmer) with the T7 RNA Polymerase. Reaction incubated for 6 h at 37°C. cRNA was purified using RNeasy Mini Kit (Qiagen). cRNA was then fragmented using the fragmentation buffer. Further processing of probes was performed according to the recommendations of the Expression Analysis Technical Manual (Affymetrix).
Hybridization protocol
Chip Type: MOE430A; Chip Lot: 3001946; Protocol: EukGE-WS2; Wash A1 Temperature (C): 25; Number of Wash A1 Cycles: 10; Mixes per Wash A1 Cycle: 2; Wash B Temperature (C): 50; Number of Wash B Cycles: 4; Mixes per Wash B Cycle: 15; Stain Temperature (C): 25; First Stain Time (seconds): 600; Wash A2 Temperature (C): 25; Number of Wash A2 Cycles: 10; Mixes per Wash A2 Cycle: 4; Second Stain Time (seconds): 600; Third Stain Time (seconds): 600; Wash A3 Temperature (C): 30; Number of Wash A3 Cycles: 15; Mixes per Wash A3 Cycle: 4; Holding Temperature (C): 25; Station: 1; Module: 2.
Scan protocol
Pixel Size: 3; Filter: 570; Number of Scans: 2; Scanner Type: HP.
Description
cRNA labelling, array hybridisation, washing and scanning performed at the Microarray Facility of the Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany.
Data processing
Scanned GeneChip *.DAT files were processed by the Affymetrix Microarray Suite 5.0.1 analysis software. Scaling: All probe sets, TGT Value: 200; Normalisation: All probe sets.
