Genepix pro v6

Microarray data extraction was performed as previously described (Gerlach et al., 2014; Kilcoyne, Gerlach, Kane, & Joshi, 2012). In brief, GenePix Pro v6.1.0.4 (Molecular Devices, Berkshire, UK) was used to extract raw intensity values from image files using a proprietary *.gal file which enabled the identification of 230 μm printed lectin spots using adaptive diameter (70%–130%) circular alignment. The data were then exported as text to Excel (version 2010, Microsoft). Local background‐corrected median feature intensity data (F633median‐B633) values were selected, and the median of six replicate spots per subarray was handled as a single data point for graphical and statistical analysis. Binding intensity data are represented in bar charts as the mean intensity with single standard deviation of all like experimental replicates.

Microarrays were washed sequentially with 0.2% (w/v) SDS buffer, PBS containing 0.1M sodium borohydride, 0.2% SDS (w/v), water and ethanol (100%). Microarray hybridizations were done on a HS48000 hybridization station (Tecan Ltd., Switzerland), using an automated program and 0.1–4 X SSC/0.1–0.2% (w/v) SDS buffer solutions as recommended by the manufacturer.Slides were scanned on an Agilent BA scanner and two digital images were obtained for each array, which were overlaid using the manufacturer’s software. Images were exported to and analyzed using GenePix® Pro v6.0 software (Molecular Devices LLC, USA) and the median of duplicate spots was calculated for each image and raw data were expressed as a log ratio of Cy5/Cy3 (sample/control probe). Intra- and inter-array global LOWESS normalization was done in J- Express Pro software (MolMine, Norway) and ratios were then used to rank genes and calculate fold changes. A False Discovery Rate (FDR) limit of ≤ 5% was applied to the gene lists and genes with an FDR above this cut-off were excluded from analysis [11 ].

Standard experimental procedures for high-density microarrays have been previously described (28 (link)). In summary, the HuProt microarray from CDI Labs consisted of approximately 21,000 proteins. The microarray was retrieved from −80 °C storage and blocked with bovine serum albumin (BSA, 9048-46-8, Sigma) for 1.5 h. Subsequently, it was incubated with plasma samples at a 1:1000-fold ratio for 1 h. After washing with 0.1% phosphate buffered solution-tween 20, the plates were incubated with Alexa fluor 647 goat anti-human IgG (109-605-003, Jackson) (diluted in 5% BSA) and scanned using GenePix 4300A (141095, Molecular Devices) with a 635 nm excitation laser. The signal intensities of lgG for each protein were quantified using GenePix Pro v.6.0 software (Molecular Devices) (https://www.moleculardevices.com/products/additional-products/genepix-microarray-systems-scanners). In the verification phase, candidate AAbs identified during the discovery phase were selected and printed onto 2 × 7 sub-arrays for an aNSCLC-focused microarray. The experimental procedures of the aNSCLC-focused microarray were similar to the high-density microarray, except for blocking and dilution buffer, which was 3% BSA.