Activity of TNF- α will be determined by enzyme-linked immunosorbent assay according to the manufacturer’s recommendations (R&D Systems, Inc.).
Summary of assay procedure74:
1. prepare reagents, standard curve dilutions, and samples as directed by the previous sections.
2. Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, reseal.
3. Add 50uL of Assay Diluent RD1-41 to the center of each well.
4. Add 50uL of Standard, Control or sample* per well. Mix by gently tapping the plate frame for one minute. Cover with the adhesive strip provided. Incubate for 2 hours at room temperature. Plate layouts are provided to record standards and samples assays.
5. Aspiration each well and wash, repeating the process four times for a total of five washes.
Wash by filling each well with Wash Buffer (400uL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
6. Add 100 uL of rat TNF-α Conjugate to each well. Cover with a new adhesive strip.
Incubate for 2 hours at room temperature.
7. Repeat the aspiration/wash as step 5.
8. Add 100 uL of substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light.
9. Add 100 uL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm. If the wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm.
This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
C. Assessment of IL-1β
Activity of IL-1β will be determined by enzyme-linked immunosorbent assay according to the manufacturer’s recommendations (R&D Systems, Inc.).
Summary of assay procedure74:
1. prepare reagents, standard curve dilutions, and samples as directed by the previous sections.
2. Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, reseal.
3. Add 50uL of Assay Diluent RD1-21 to each well.
4. Add 50uL of Standard, Control or sample* per well. Mix by gently tapping the plate frame for one minute. Cover with the adhesive strip provided. Incubate for 2 hours at room temperature. Plate layouts are provided to record standards and samples assays.
5. Aspiration each well and wash, repeating the process four times for a total of five washes.
Wash by filling each well with Wash Buffer (400uL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
6. Add 100 uL of rat IL-1β Conjugate to each well. Cover with a new adhesive strip.
Incubate for 2 hours at room temperature.
7. Repeat the aspiration/wash as step 5.
8. Add 100 uL of substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light.
9. Add 100 uL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm. If the wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate.
Readings made directly at 450 nm without correction may be higher and less accurate.
D. Assessment of IL-6
Activity of IL-6 will be determined by assessing the 72-hour proliferation of the IL-6-dependent murine hybridoma 7TD1 stimulated by serial dilutions of plasma or supernatant, as described previously.
Summary of assay procedure76:
1. Bring all reagents to room temperature. Prepare reagents and samples as instructed. Return unused components to storage temperature as indicated in the instructions.
2. Add 50uL of Assay Diluent RD1-54 to each well.
3. Add 50uL of Standard, Control or sample* to each well. Tap plate gently for one minute.
Cover the plate and incubate for 2 hours at room temperature.
4. Aspirate and wash each well for five times.
5. Add 100 uL of rat IL-6 Conjugate to each well. Cover the plate and incubate for 2 hours at room temperature.
6. Aspirate and wash each well for five times.
7. Add 100 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light.
8. Add 100 uL of Stop Solution to each well.
9. Read Optical, Density at 450 nm (correction wavelength set at 540 nm or 570 nm) E. Assessment of IL-10
Concentrations of IL-10 will be determined by enzyme-linked immunosorbent assay according to the manufacturer’s recommendations (Pharmingen, San Diego, CA).
Summary of assay procedure77:
1. Bring all reagents to room temperature. Prepare reagents and samples as instructed. Return unused components to storage temperature as indicated in the instructions.
2. Add 50uL of Assay Diluent RD1-21 to each well.
3. Add 50uL of Standard, Control or sample* to each well. Tap plate gently for one minute.
Cover the plate and incubate for 2 hours at room temperature.
4. Aspirate and wash each well for five times.
5. Add 100 uL of rat IL-10 Conjugate to each well. Cover the plate and incubate for 2 hours at room temperature.
6. Aspirate and wash each well for five times.
7. Add 100 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light.
8. Add 100 uL of Stop Solution to each well.
9. Read Optical, Density at 450 nm (correction wavelength set at 540 nm or 570 nm) [Animal Liver Tissue Harvest]
The harvest of rat liver was performed through a midline ventral incision from pubis to xiphoid process. One segment (weight > 1 g) of middle lobe of liver is excised and hemostasis is done by ligation with 3-0 silk at the proximal part of middle lobe. The liver sample will be frozen rapidly in liquid nitrogen. The tissue will be kept in a -70oC freezer until microarray analysis.
[Microarray & Technique81,82] A. Introduction
Expression of liver RNA was analysed by microarray technique using GeneChip®Probe Arrays (Affymetrix, Inc.). GeneChip probe arrays are manufactured using technology that
combines photolithography and combinatorial chemistry. Up to 1.3 million different oligonucleotide probes are synthesized on each array. Each oligonucleotide is located in a specific area on the array called a probe cell. Each probe cell contains hundreds of thousands to millions of copies of a given oligonucleotide.
During the laboratory procedure described in this manual, biotin-labeled RNA or DNA fragments referred to as the “target” are hybridized to the probe array. The hybridized probe array is stained with streptavidin phycoerythrin conjugate and scanned by the GeneChip® Scanner 3000, or the GeneArray® Scanner. The amount of light emitted at 570 nm is proportional to the bound target at each location on the probe array.
B. GeneChip® Expression Analysis 1. RNA Preparation
Total RNA was isolated from rat liver tissue samples and electrophoresed on a 1%
agarose-formaldehyde gel to determine the integrity of the RNA preparation. All RNA samples should meet assay quality standards to ensure the highest quality RNA is hybridized to the gene expression arrays. A 260/280 absorbance reading should be obtained for both total RNA and biotinylated cRNA. Acceptable A260/280 ratios fall in the range of 1.8 to 2.1. Ratios below 1.8 indicate possible protein contamination. Ratios above 2.1 indicate presence of degraded RNA, runcated cRNA transcripts, and/or excess free nucleotides. Then double-stranded cDNA is synthesized from total RNA isolated from rats’ liver tissue. An in vitro transcription (IVT) reaction is then done to produce biotin-labeled cRNA from the cDNA. The cRNA is fragmented before hybridization. The use of gel electrophoresis will aid in quality control following the sample from step to step in the assay and hybridization protocol. Gel electrophoresis can be performed after cDNA synthesis (if using poly-A mRNA as starting material), after cRNA synthesis, and after fragmentation. This will be helpful in estimating quantity and size distribution of the labeled sample. During this phase of technical evaluation, cRNA yield from a standard total RNA sample is another simple and effective method to assess consistency.
2. Gene microarray hybridization
A hybridization cocktail is prepared, including the fragmented target, probe array controls, BSA, and herring sperm DNA. It is then hybridized to the probe array during a 16-hour incubation. The hybridization process is described in the respective sections for the different probe array types.
3. Fluidics Station Setup
Specific experimental information is defined using Affymetrix® Microarray Suite or GeneChip Operating Software (GCOS) on a PC-compatible workstation. The probe array type, sample description, and comments are entered and saved with a unique experiment name. The fluidics station is then prepared for use by priming with the appropriate buffers.
4. Probe Array Washing and Staining
Immediately following hybridization, the probe array undergoes an automated washing and staining protocol on the fluidics station.
5. Probe Array Scan
Once the probe array has been hybridized, washed, and stained, it is scanned. Each workstation running Affymetrix Microarray Suite or GCOS can control one scanner. The software defines the probe cells and computes an intensity for each cell. Each complete probe array image is stored in a separate data file identified by the experiment name and is saved with a data image file (.dat) extension.
6. Data analysis
The .dat image is analyzed for probe intensities; results are reported in tabular and graphical formats. Information on data analysis is provided in the enclosed GeneChip®
Expression Analysis: Data Analysis Fundamentals booklet (P/N 701190).