Mice were immunized intradermally with 100 µL of Incomplete Freund’s Adjuvant (IFA) containing 200 µg of bovine CII (Elastin Products) and 200 µg of inactivated Mycobacterium tuberculosis (H37Ra; Difco) on days 0 and 21. Each experimental group consisted of 8 mice that were treated daily throughout the entire experiment, i.e. day 0 of the first injection through sacrifice on day 35, with one of five interventions: no injection, PBS (vehicle control), 1 mg/kg Cl-amidine, 10 mg/kg Cl-amidine or 50 mg/kg Cl-amidine. All injections were given i.p. in a standard volume of 200 μL containing the appropriate concentration of Cl-amidine. Doses were calculated for the average weight of the group (20 g). At day 35, all animals were sacrificed by anesthesia with 2,2,2-tribromoethanol and cervical dislocation. One mouse in the 1 mg/kg treatment group died on day 8 of the experiment of causes unrelated to treatment.
Growth protocol
Male DBA/1J mice were obtained from Jackson Laboratories and were between 6 and 8 weeks of age when experiments were initiated.
Extracted molecule
protein
Extraction protocol
At the end of the injection courses (day 35), the mice were bled for their plasma.
Label
Cy3
Label protocol
After washing with phosphate-buffered saline (PBS)/3% fetal bovine serum/0.1% Tween 20, reactive antibodies were detected using Cy3-conjugated goat-anti-mouse IgG/IgM secondary antibody (diluted 1:4,000 in PBS/3% fetal bovine serum; Jackson Immunoresearch). After incubating for 45 minutes with the secondary antibody, the slides were washed with washing solution (PBS/3 % fetal bovine serum/0.1% Tween 20
Hybridization protocol
Synovial antigen arrays were produced using a robotic microarrayer to print peptides and proteins on ArrayIt SuperEpoxy microscope slides (TeleChem International, Sunnyvale, CA). On each slide, antigens are printed at 0.2 mg/ml in four replicates. Slides were then blocked overnight (phosphate-buffered saline (PBS), 3% fetal bovine serum, 0.1% Tween 20) and incubated for 1h at 4°C with individual mouse serum diluted at 1:200 in PBS/3% fetal bovine serum. Slides were washed with PBS, water-rinsed, and spun dry.
Scan protocol
Slides were scanned to determine pixel intensities for each antigen feature (Genepix 4000B; Molecular Devices, Sunnyvale, CA, USA). Genepix Pro 5.0 software (Axon Instruments) at 532 nm was used to detect IgG and IgM reactivity. Images were saved as tiff files.
Description
1:200 dilution in PBS with 3% Fetal Bovine Serum
Data processing
GenePix Pro 6.0 software (Axon Instruments) was used to determine the net median pixel intensities for each antigen feature (each value is background-corrected by removing the background around each spotted protein). The median intensities for each protein were centered around 300 and log-scaled. A level of 10 digital fluorescence intensity units was set as a threshold for significant reactivity. The formula for values below 10 is log(10/300,2), and for all other values it is log(median pixel intensity/300,2). Data analysis was performed using Significance Analysis for Microarrays (SAM) software (http://www-stat.stanford.edu/~tibs/SAM) to identify antigen features with statistically significant differences in reactivities between the experimental groups. Cluster software was then used to hierarchically group the samples and antigen features on the basis of a pairwise similarity function, and TreeView software was used to display the data as a heat map (http://rana.lbl.gov/EisenSoftware.htm).
Cl-amidine treatment reduces autoantibody response in collagen-induced arthritis
Data table header descriptions
ID_REF
VALUE
A level of 10 digital fluorescence intensity units was set as a threshold for significant reactivity. The formula for values below 10 is log(10/300,2), and for all other values it is log(median pixel intensity/300,2).
