Investigation Title Transcription profiling of mouse hippocampus treated with intracerebroventricular administration of LPS in a vehicle controlled experiment Comment[Submitted Name] NF2_LPS_MG_ICV_BV2 Experimental Design stimulus_or_stress_design disease_state_design transcription profiling by array Experimental Design Term Source REF mo mo EFO Comment[ArrayExpressReleaseDate] 2005-10-01 Comment[AEMIAMESCORE] 5 Comment[ArrayExpressAccession] E-MEXP-472 Comment[MAGETAB TimeStamp_Version] 2011-06-28 22:11:39 Last Changed Rev: 14857 Experimental Factor Name time disease state material_type Experimental Factor Type time disease_state material_type Experimental Factor Term Source REF Person Last Name Poerzgen Person First Name Peter Person Mid Initials Person Email poerzgen@fastmail.fm Person Phone +1 808 393 6117 Person Fax Person Address Ottiliavej 9 Person Affiliation Molecular Neurobiology, Lundbeck A/S Person Roles submitter Person Roles Term Source REF Quality Control Type dye_swap_quality_control Quality Control Term Source REF The MGED Ontology Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2005-10-01 PubMed ID Publication DOI Publication Author List Publication Title Publication Status Publication Status Term Source REF Experiment Description Effect of icv (intracerebroventricular) administration of LPS in mice in a vehicle controlled experiment. After administrating LPS icv, the hippocampus is dissected out and inflammatory gene inductions are chip analyzed Protocol Name P-MEXP-10434 P-MEXP-10433 P-MEXP-11049 P-MEXP-10432 P-MEXP-11050 P-MEXP-10435 P-MEXP-10431 P-MEXP-10430 P-MEXP-11048 P-MEXP-8285 P-MEXP-10436 P-MEXP-11042 P-MEXP-8287 P-MEXP-8350 P-MEXP-11045 P-MEXP-11046 P-MEXP-8288 P-MEXP-11047 P-MEXP-8314 P-MEXP-14771 Protocol Type specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action grow grow grow pool nucleic_acid_extraction nucleic_acid_extraction labeling labeling labeling labeling hybridization hybridization image_acquisition bioassay_data_transformation Protocol Description Cell incubation scheme. All incubations were performed at 37 C, 5 % CO2, 95 % relative humidity. Suspended primary microglia were seeded at 3 million cells/Petri dish (Nunc, surface area 20 cm3) in 5 ml medium (DMEM, 10 % FCS). After 25 min of incubation, loosely adherent cells were removed by tapping the sides of the dish, followed by two washes in PBS. After over night incubation, cells were washed twice in PBS followed by addition of 5 ml medium (DMEM, 1% FCS). The primary microglia were then stimulated with LPS (100 ng/ml)(Salmonella abortus equi, from Biocloth (Aidenbach, Germany) for 4 hours. Then total RNA was isolated, and the supernatant was sampled for ELISA analysis of cytokine production. All medium constituents were purchased from Invitrogen (Taastrup, Denmark). Mice (C57bl/6j) from Harlan (Horst, Holland) were anesthetized with sevoflurane and fixed in a homemade frame where after LPS or vehicle was administered i.c.v at a dose of 2.25 microgram/brain in 2.5 microL (over 1.5 minutes) into the right ventricle through a 5/8 inches 25-gauge needle connected to a 100 microL Hamilton syringe (Martinsried, Germany) which was mounted in a micro pump. The electrolyte composition of the vehicle was 140 mM NaCl, 3 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 1.2 mM Na2HPO4, adjusted to pH 7.4. Mice were sacrificed either 16 h after the i.c.v injection by cervical dislocation, and the hippocampus was dissected free and stored in RNAlater (Invitrogen) until RNA extraction. 20 hours before an experiment cells were washed and detached as described above. The cells were counted in a counting chamber (Fast Read, ISL) and 5*106 cells for the 4-hour time point experiment and 2*106 cells for the 16-hour time point experiment were transferred to 145 cm2 Petri dishes (NUNC) containing 20 ml of culture medium. Two dishes per condition (ctrl, 4hr/16hr and LPS stimulated, 4hr/16hr) were set up.
Just before an experiment cells were washed once, gently, with pre-warmed PBS and 20 ml of culture medium with only 1% FBS and antibiotics was added. Immediately after, 100 ml of a 20 mg/ml lipopolysaccharide, LPS, stock (Salmonella abortus equip.) diluted in PBS was added to half of the dishes getting a final concentration of 100 ng/ml LPS. Mice (C57bl/6j) from Harlan (Horst, Holland) were anesthetized with sevoflurane and fixed in a homemade frame where after LPS or vehicle was administered i.c.v at a dose of 2.25 microgram/brain in 2.5 microL (over 1.5 minutes) into the right ventricle through a 5/8 inches 25-gauge needle connected to a 100 microL Hamilton syringe (Martinsried, Germany) which was mounted in a micro pump. The electrolyte composition of the vehicle was 140 mM NaCl, 3 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 1.2 mM Na2HPO4, adjusted to pH 7.4. Mice were sacrificed either 4 h after the i.c.v injection by cervical dislocation, and the hippocampus was dissected free and stored in RNAlater (Invitrogen) until RNA extraction. 20 hours before an experiment cells were washed and detached as described above. The cells were counted in a counting chamber (Fast Read, ISL) and 5*106 cells for the 4-hour time point experiment and 2*106 cells for the 16-hour time point experiment were transferred to 145 cm2 Petri dishes (NUNC) containing 20 ml of culture medium. Two dishes per condition (ctrl, 4hr/16hr and LPS stimulated, 4hr/16hr) were set up.
Just before an experiment cells were washed once, gently, with pre-warmed PBS and 20 ml of culture medium with only 1% FBS and antibiotics was added. Immediately after, 100 ml of a 20 mg/ml lipopolysaccharide, LPS, stock (Salmonella abortus equip.) diluted in PBS was added to half of the dishes getting a final concentration of 100 ng/ml LPS. Cell incubation scheme. All incubations were performed at 37 C, 5 % CO2, 95 % relative humidity. Suspended primary microglia were seeded at 3 million cells/Petri dish (Nunc, surface area 20 cm3) in 5 ml medium (DMEM, 10 % FCS). After 25 min of incubation, loosely adherent cells were removed by tapping the sides of the dish, followed by two washes in PBS. After over night incubation, cells were washed twice in PBS followed by addition of 5 ml medium (DMEM, 1% FCS). The primary microglia were then stimulated with LPS (100 ng/ml)(Salmonella abortus equi, from Biocloth (Aidenbach, Germany) for 16 hours. Then total RNA was isolated, and the supernatant was sampled for ELISA analysis of cytokine production. All medium constituents were purchased from Invitrogen (Taastrup, Denmark). All experimental procedures were carried out in accordance with the directives of the Danish National Committee on Animal Research Ethics and the European Communities Council Directive #86/609 for care of laboratory animals. Preparation of primary murine microglia culture. Pregnant C57bL/6J mice were purchased from Harland (Horst, Holland). Microglia cultures were prepared as initially described by Guillian & Baker as follows: Pups (1-3 days postpartum) were decapitated and the cerebra were transferred to DMEM (with 20 % heat inactivated FBS, supplemented with antibiotics (penicillin 100 U/ml, streptomycin 100 mg/ml). This medium was used for all work related to primary microgila (PM), only the FBS concentration was varied. Following removal of meninges, brains were triturated with a 10 ml pipette to obtain a homogeneous cell suspension that was subsequently passed though a 70 mm cell strainer (Falcon) into 50 ml test tubes (Nunc). The cell suspension was plated at a density of 3 brains/185 cm2 flask and cultured undisturbed for 7 days. Then, new medium with reduced serum concentration (10 % FBS) was added. After 7 additional days of culturing, microglial cells were selectively detached by shaking (300 rpm, 1 h). Suspended microglia were pelleted at 180 x g, before being seeded. Purity was always > 95 % as determined by routine staining with FITC-labeled lectin from Bandeiraea simplicifolia BS-I (Molecular probes/Invitrogen, Taastrup, Denmark). All medium constituents were purchased from Invitrogen (Taastrup, Denmark). C57bl/6j male mice, 3 months of age, were purchased from Harlan (Horst, Holland) and housed 5 pr cage at 20-24 degrees celcius with food and water adlibitum in a 12 hours day/night cyclus. Animals were allowed to acclimatize for one week before use. The murine microglial cell line BV-2, kindly provided by E. Blasi, Perugia, (Blasi et al., 1990), was maintained in culture medium (RPMI1640 Medium + glutamax TM1 (Gibco Invitrogen corp.) supplemented with 10% heat-inactivated fetal bovine serum, FBS, (Gibco Invitrogen corp.) and antibiotics, P/S, (penicillin 100 U/mL, streptomycin 100 mg/mL (Gibco Invitrogen corp.)), and kept in 75 cm2 culture flasks (NUNC) in a humidified incubator (Heraeus BBD 6220, Kendro Laboratory products) at 37 C, 5 % CO2 and 95 % relative humidity.
Every 2nd day, when reaching a confluence of 80-90%, cells were transferred to a laminar airflow, LAF, bench (Heto-Holten, model 1.2), washed once with 10 ml of pre-warmed phosphate buffered saline, PBS, (Gibco Invitrogen corp.) and detached in 10 ml of pre-warmed culture medium using a cell scraber (NUNC). One ml of triturated cell suspension was then transferred to a new 75 cm2 culture flask (NUNC) containing 9 ml of fresh pre-warmed medium and placed in incubator. Each sample consists of one 185 cm2 bottle containing 10-15 million astrocytes (confluency 90-95%). Routinely we isolated 20-30ug of total RNA from one culture flask. Of this, 10ug were used for the amplification reaction. The resulting cRNA was split in two 10ug aliquots and labeled in parallel with either Cy3 or Cy5 dye-esters. This means, that the material isolated from one culture flask was used for a complete dye-swap experiment, no pooling occurred. The remaining total RNA was used for verification of array results (e.g. qPCR). RNA preparation and labeling was carried out as described in protocols on Joseph DeRisi's website (www.microarrays.org/protocols.html), and protocols available from MWG-Biotech (www.MWG-biotech.com). In brief: 5-10 ug of total RNA were reverse transcribed using a [(dT24)T7promotor]65 primer for the 1st strand synthesis, followed by a 2nd strand synthesis according to the manufactures protocol (cDNA Synthesis System, Roche Diagnostics, Cat. No 1117831). The resulting (double stranded) dsDNA was purified using Roche's High Pure RNA Tissue Kit (Cat. No 2033674). The purified dsDNA was amplified using Ambion's MEGAscriptT7 kit (Cat. No 1334) and included the incorporation of aminoallyl-modified UTP (aa-UTP). The reaction mix contained the following concentrations of nucleotides: 7.3 mM ATP, CTP and GTP, 3.6 mM UTP and 4.7 mM aa-UTP (Sigma, Cat. No A5660) and was incubated for 16 h at 37 C. The amplified RNA was purified using Roche's High Pure RNA Tissue Kit and, subsequently, its concentration and purity was controlled by spectro-photometric analysis. We usually obtained 30-50 ug of amplified cRNA from a starting material of 5 ug total RNA. Routinely we isolated 20-30ug of total RNA from one culture flask. RNA preparation and labeling was carried out as described in protocols on Joseph DeRisi's website (www.microarrays.org/protocols.html), and protocols available from MWG-Biotech (www.MWG-biotech.com).cDNA synthesis and labelling 15 mg of total RNA were reverse transcribed using random hexamere and dT16 primers and Superscript II reverse transcriptase (Invitrogen), incorporating amino-allyl dUTP into the 1st-strand cDNA.After the cDNA synthesis, the remaining RNA was hydrolyzed and cleaned up withMicrocon-30 spin filters (Millipore). 10ug of the amino-allyl modified cRNA were coupled to the N-hydroxy-succinimidyl esters of the Cy3-dye following Joe de Risi's protocol (http://www.microarrays.org/pdfs/amino-allyl-protocol.pdf). The dye-esters were from Amersham (Cy3- and Cy5 mono-reactive dye, PA23001 and PA25001). The labelled cRNA was then purified from the excess dye using Qiagen's RNeasy Mini columns and their 'RNA clean-up' protocol. Finally, in order to perform a 'dye-swap' experiment, the Cy3-labelled cRNA sample was split in two equal aliquots and combined with the Cy-5-labelled samples of a second cRNA preparation (e.g. Cy3-vehicle versus Cy5-CCM and Cy5-vehicle versus Cy3-CCM). 10ug of the amino-allyl modified cRNA were coupled to the N-hydroxy-succinimidyl esters of the Cy5-dye following Joe de Risi's protocol (http://www.microarrays.org/pdfs/amino-allyl-protocol.pdf). The dye-esters were from Amersham (Cy3- and Cy5 mono-reactive dye, PA23001 and PA25001). The labelled cRNA was then purified from the excess dye using Qiagen's RNeasy Mini columns and their 'RNA clean-up' protocol. Finally, in order to perform a 'dye-swap' experiment, the Cy5-labelled cRNA sample was split in two equal aliquots and combined with the Cy-3-labelled samples of a second cRNA preparation (e.g. Cy3-vehicle versus Cy5-CCM and Cy5-vehicle versus Cy3-CCM). Half of the amino-allyl modified cDNA were coupled to the N-hydroxy-succinimidyl esters of the Cy3-dye following Joe de Risi's protocol (http://www.microarrays.org/pdfs/amino-allyl-protocol.pdf). The dye-esters were from Amersham (Cy3- and Cy5 mono-reactive dye, PA23001 and PA25001). The labelled cDNA was then purified from the excess dye with the QiaQuick (Qiagen) PCR Purification columns work. Finally, in order to perform a 'dye-swap' experiment, the Cy3-labelled cDNA sample was split in two equal aliquots and combined with the Cy-5-labelled samples of a second cDNA preparation. Half of the amino-allyl modified cDNA were coupled to the N-hydroxy-succinimidyl esters of the Cy5-dye following Joe de Risi's protocol (http://www.microarrays.org/pdfs/amino-allyl-protocol.pdf). The dye-esters were from Amersham (Cy3- and Cy5 mono-reactive dye, PA23001 and PA25001). The labelled cDNA was then purified from the excess dye with the QiaQuick (Qiagen) PCR Purification columns work. Finally, in order to perform a 'dye-swap' experiment, the Cy5-labelled cDNA sample was split in two equal aliquots and combined with the Cy-3-labelled samples of a second cDNA preparation. Before hybridisation, the combined and labelled cRNA samples were submitted to a mild alkaline fractionation step at 94°C for 15 min in 4 mM Tris Acetate (pH=8.1), 3 mM MgAc and 10 mM KAc, quickly followed by re-buffering and concentrating using Microcon YM-10 spin filters (Millipore) and a speed-vac centrifuge. The cy-labelled cRNAs were dissolved in Gene Frame Hybridization buffer (MWG Biotech, Ebersberg, Germany, Cat. No. 180-200000), denatured for 5 min at 90°C, before incubation on the microarray slides for 16 h at 42°C. Before hybridisation the Cy-labelled cDNAs was combined in Gene Frame Hybridization buffer (MWG Biotech, Ebersberg, Germany, Cat. No. 180-200000). The hybridization mixture was denatured for 5 min before incubation on the slides for 16 h at 42 C. Hybridisation was followed by three washing steps of increasing stringency: 2x SSC, 0.1% SDS followed by 1x SSC, 0.01% SDS and 0.5x SSC (all solutions were preheated to 30°C). Finally, each slide was spun dry and scanned in a 428 Affymetrix confocal laser scanner at three different intensities (=photo multiplier gains, usually between 35-60). Other scanner parameters were X=1.5mm; Y=16mm; width=22mm; height=48.5mm. Images were saved as Tiff-files and further analysed with ImaGene4.0 software (Biodiscovery, see normalisation protocol). The microarrays were analysed using ImaGene 4.2 (BioDiscovery Inc.) for spot location, array alignment and background subtraction. Signal intensities for individual spots were adjusted for local background. Microsoft Excel was used for further statistical analysis of the ImaGene output files. E.g. Cy3/Cy5 ratio normalization was carried out by multiplying each ratio value with a scaling factor, which was defined as the ratio of the overall signal intensity of the Cy5 versus Cy3 channel as described in Knudsen, S. 2002. A Biologist's Guide to Analysis of cDNA Microarray Data. New York, Wiley Interscience. Each microarray experiment was performed at least twice independently. To further account for bias introduced by dye bleaching or labelling, each experiment was carried out as dye-swap experiment with the resulting ratio value being the arithmetical mean from two slides of opposite labelled sample pairs. Protocol Parameters Amplification;Extracted product; Extracted product;Amplification; Amount of nucleic acid labeled;Label used;Amplification; Amplification;Label used;Amount of nucleic acid labeled; Amplification;Label used; Label used;Amplification; Chamber type;temperature;Volume;time;Quantity of label target used; Quantity of label target used;Chamber type;temperature; Protocol Hardware Scanning hardware Protocol Software Default scanner software Protocol Contact Protocol Term Source REF mo mo mo mo mo mo mo mo mo mo SDRF File E-MEXP-472.sdrf.txt Term Source Name NCI_thesaurus ncbitax mo ArrayExpress The MGED Ontology mo EFO Term Source File ncithesaurus.obo.alt http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ Term Source Version