Peritoneal exudate cells obtained by lavage, and single cell suspensions obtained by collagenase digestion of tissue capsules, were separated on the basis of size/granularity and EGFP fluorescence.
Growth protocol
Sterile foreign objects were inserted into the peritoneal cavities of MacGreen mice (in which the Csf1r promoter directs myeloid-specific EGFP expression). At various time-points post-surgery (days 2, 4, 7, 14), mice were euthanased, and peritoneal exudate cells collecteded by lavage. Peritoneal exudate cells from MacGreen mice that had not received implants were used as controls (day 0; 'unstimulated'). Free-floating objects encapsulated with tissue were removed from the peritoneal cavities of different mice at days 7, 14, 21, 28.
Extracted molecule
total RNA
Extraction protocol
Total RNA from pooled FACS-sorted EGFPhi peritoneal exudate and tissue capsule cells was obtained using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA quality was analysed for quality by Bioanalyser (Agilent, Santa Clara, CA) and quantity by spectrophotometer (ND-1000; Thermo Scientific, Waltham, MA).
Label
biotin
Label protocol
Biotin-labeled amplified mRNA (aRNA) was synthesized with the Illumina RNA Amplification Kit (Ambion, Austin, TX).
Hybridization protocol
cDNA was synthesized using Illumina RNA Amplification Kit and hybridized to Illumina Mouse-6 v1.1 BeadChip microarrays according to manufacturer's instructions.
Scan protocol
Bead array (Illumina)
Description
replicate 2 4112157060_B
Data processing
Illumina BeadStudio extracted gene expression intensities were background corrected and quantile normalized using Lumi v2.10 (Bioconductor v2.18, R v2.15.3).
Submission date
Dec 06, 2013
Last update date
Sep 17, 2014
Contact name
Othmar Korn
Organization name
Australian Institute for Bioengineering and Nanotechnology
