|
| Status |
Public on Aug 13, 2020 |
| Title |
BMDM_wt_HemeAlb200LPS_a |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
BMDM
|
| Organism |
Mus musculus |
| Characteristics |
treatment: no treatment
incubation time: 0h
|
| Treatment protocol |
Macrophages were exposed to heme-albumin complexes or albumin alone (200 mcM) for four hours before challenge with LPS (10 ng/ml)
|
| Growth protocol |
Bone marrow cells were obtained from the femurs and tibias of 8- to 10-week-old mice. The bone marrow murine stem cells were expanded and differentiated in RPMI medium supplemented with 10% FCS in the presence of recombinant mouse M-CSF (PeproTech 315-02-100UG, lot 0914245) at 10 ng/ml for 7 days
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After washing and lysing the cells with Buffer RLT, total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Basel, Switzerland) according to the manufacturer’s instructions, including an on-column DNA digestion step (RNase-Free DNase Set; QIAGEN). To ensure that only high quality RNA (RIN >7.0) was used for gene expression analysis, each RNA sample was checked on a RNA Nanochip with a Bioanalyzer 2100 (Agilent Technologies, Basel, Switzerland). RNA was quantified spectrophotometrically with a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA).
|
| Label |
Cy3 (green signal)
|
| Label protocol |
Fluorescently labeled cRNA was generated from 500 ng total RNA with the Quick Amp Labeling Kit (Agilent) according to Agilent’s Two-Color Microarray-Based Gene Expression Analysis Protocol Version 5.7, March 2008 (Agilent publication number G4140-90050). In short, total RNA was reverse transcripted to cDNA using T7 Promotor Primer and MMLV-RT. Then cDNA was converted to cRNA using T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3- or cyanine 5-labeled CTP.
|
| |
|
| Channel 2 |
| Source name |
BMDM
|
| Organism |
Mus musculus |
| Characteristics |
treatment: heme-albumin (4h, 200 mcM) + LPS (4h)
incubation time: 4h + 4h
|
| Treatment protocol |
Macrophages were exposed to heme-albumin complexes or albumin alone (200 mcM) for four hours before challenge with LPS (10 ng/ml)
|
| Growth protocol |
Bone marrow cells were obtained from the femurs and tibias of 8- to 10-week-old mice. The bone marrow murine stem cells were expanded and differentiated in RPMI medium supplemented with 10% FCS in the presence of recombinant mouse M-CSF (PeproTech 315-02-100UG, lot 0914245) at 10 ng/ml for 7 days
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After washing and lysing the cells with Buffer RLT, total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Basel, Switzerland) according to the manufacturer’s instructions, including an on-column DNA digestion step (RNase-Free DNase Set; QIAGEN). To ensure that only high quality RNA (RIN >7.0) was used for gene expression analysis, each RNA sample was checked on a RNA Nanochip with a Bioanalyzer 2100 (Agilent Technologies, Basel, Switzerland). RNA was quantified spectrophotometrically with a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA).
|
| Label |
Cy5 (red signal)
|
| Label protocol |
Fluorescently labeled cRNA was generated from 500 ng total RNA with the Quick Amp Labeling Kit (Agilent) according to Agilent’s Two-Color Microarray-Based Gene Expression Analysis Protocol Version 5.7, March 2008 (Agilent publication number G4140-90050). In short, total RNA was reverse transcripted to cDNA using T7 Promotor Primer and MMLV-RT. Then cDNA was converted to cRNA using T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3- or cyanine 5-labeled CTP.
|
| |
|
| |
| Hybridization protocol |
After purification of amplified cRNA according to the QIAgen RNeasy purification protocol (QIAGEN) 825 ng of Cy3 and Cy5 labeled cRNA were competitively hybridized for 17 hours at 65° C in a hybridization oven (Agilent G2545A) set to 10 rpm in GEx Hybridization buffer HI-RPM according to the manufacturer's protocol (Agilent’s Two-Color Microarray-Based Gene Expression Analysis). Washing was performed according to the recommended protocol including the Stabilization and Drying Solution step.
|
| Scan protocol |
Array slides were XDR scanned at 5 mcm resolution on a Agilent Microarray High-Resolution C Scanner (G2505C) with 100% PMT and 10% for lower intensity.
|
| Description |
Nrf2+/+ Bone marrow derived macrophages pretreated with Albumin plus 200 mcM Heme for 4 hours and then with LPS for 4 hours
|
| Data processing |
Images were auto gridded, analyzed and data extracted using Agilent Feature Extraction Software Version 10.5.1.1. Spot values were normalized using the default linear-lowess normalization. To identify differentially expressed features, calculated log10 ratio values (Cy5/Cy3) were imported into JMP Genomics 8.0 without further normalization and the built-in Basic Expression Workflow (ANOVA, LSMeans Differences) was performed.
We imported the calculated and lowess-normalized log10 ratios of the Feature Extraction result text files directly into JMP Genomis 8.0 and performed the statistical analysis without further normalization.
For the statistical analysis of our array experiments we imported the log10 ratios of the Feature Extraction result text files into JMP Genomis 8.0. As Feature Extraction Software does already a linear-lowess normalization, we didn't perform any further normalization. That means that our normalized data are already included in the Agilent Feature Extraction Files, which have been uploaded with this Metadata Worksheet. Any further data processing, i.e. filtering for x-fold over- or underexpression of significant features, we performed after the ANOVA analysis.
|
| |
|
| Submission date |
Mar 01, 2020 |
| Last update date |
Aug 14, 2020 |
| Contact name |
Marc Pfefferle |
| E-mail(s) |
marc.pfefferle@uzh.ch
|
| Organization name |
University hospital Zürich (USZ)
|
| Street address |
Wagistrasse 12
|
| City |
Schlieren |
| State/province |
Schweiz |
| ZIP/Postal code |
8952 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL10333 |
| Series (2) |
| GSE145244 |
Erythrophagocytosis drives anti-inflammatory programming of liver macrophages |
| GSE146152 |
Erythrophagocytosis drives anti-inflammatory programming of liver macrophages [Agilent] |
|
