Innoscan 710 microarray scanner

The genomic DNA was extracted from CD123- cells from UCART123 treated mice in PDX-3 experiment. The quality of genomic DNA was tested by NanoDrop 2000 (Thermo Scientific). 1.0 µg genomic DNA from CD123 + cells from vehicle-treated mice, which is used as reference, was labeled with Cy3 and Cy5 fluorescent dyes, respectively, by using CytoSure^TM HT Genomic DNA Labelling Kit (Cat# 500040, Oxford Gene Technology, UK). Labeled DNA was further purified with Clean-up columns (Cat# 500020, Oxford Gene Technology, UK) to remove unincorporated fluorescent dyes. Labeled DNA was measured the absorbance at 260 nm, 55 nm and 650 nm by NanoDrop 2000. Equal amount of labeled DNA from CD123- cells and CD123 + cells were mixed in hybridization buffer, loaded on 8x60K CytoSure ISCA + SNP array (Cat#20052, Oxford Gene Technology, UK) and hybridize at 65 °C for 22 h in the hybridization oven. After post washing, the array slide was scanned by InnoScan 710 Microarray Scanner (Innopsys, France) and further analyzed using CytoSure Interpret software version 4.8.32 (Oxford Gene Technology, UK).

Conditioned media from hDFs exposed to the different conditions were collected after 7 days of treatment and stored at −80 °C. Cytokine profiles were measured by Quantibody Human Inflammatory Array 1 (RayBiotech, QAH-INF-1-1, Peachtree Corners, GA, USA) which permitted the detection of 10 inflammation-associated cytokines, including interleukin (IL)-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-13, monocyte chemoattractant protein-1 (MCP-1), interferon (IFN)-γ and TNF-α in a single procedure. One standard glass slide was divided into 16 wells of identical cytokine antibody arrays. Each antibody, together with the positive controls, was arrayed in quadruplicate. The protein array slides spotted by specific capture antibodies were incubated with thawed media, washed, and incubated with a cocktail of biotinylated antibodies using the protocol provided by the manufacturer. The slides with bound biotin were then incubated with Cy3 equivalent dye-conjugated streptavidin. Relative fluorescent strength was detected by the InnoScan 710 microarray scanner (Innopsys, Chicago, IL, USA). The results are shown as fold change release relative to untreated cells.

The Array Comparative Genomic Hybridization (aCGH) characterization was performed using the oligonucleotide-based microarray CytoChip Cancer 8 × 60 K v2.0 for Chor-IN-1, U-CH1 and U-CH2 samples and CytoChip ISCA 8 × 60 K v2.0 for MUG-Chor1 and JHC7 samples (BlueGenome Ltd, Cambridge, UK). Both arrays consist of 60,000 60-mer oligonucleotides spaced at about 50 kb density over the full genome. Labeling and hybridization were performed following the manufacturer’s protocol. 500 ng of the DNA extracted from each sample was co-hybridized with an equal amount of reference DNA. Reference sample was chosen according to the gender of the analyzed sample (Human Genomic DNA male G1471; Human Genomic DNA female G1521–Promega, WI, USA). The analysis was performed using InnoScan 710 Microarray scanner (Innopsys, Carbonne, France) and Mapix software (Innopsys, Carbonne, France). Genomic profiles were obtained using Blufuse Multi v4.3 software (BlueGnome Ltd, Cambridge, UK).

Biotinylated glycans 1–27 were printed in replicates of six using a Scienion sciFLEXARRAYER S3 (Berlin, Germany) on streptavidin-coated glass slides^{21 (link)}. Recombinant HA was premixed with mouse anti-streptag-Alexa647 and goat-anti mouse-Alexa647 antibodies in a molar ratio of 4:2:1 in PBS-T (50 µl) for 15 min on ice. Sub-arrays (6 × 27 spots) were incubated with the premixed HA for 90 min in a humidified chamber before washing of the whole slide in 4 successive steps with PBS-T, PBS and deionized water with 5-min soak times. Arrays were dried by centrifugation and fluorescence was scanned using an Innopsys InnoScan 710 microarray scanner (Carbonne, France). The data were processed with GenePix Pro 7 (Molecular Devices, San Jose, CA) and analyzed with an in-house developed Excel macro using Excel 2016⁷² . The highest and lowest replicates were removed, and the mean and standard deviation were calculated (n = 4). Data were plotted using GraphPad Prism 7.0 (San Diego, CA).

Biotinylated glycans 1–27 were printed in replicates of six using a Scienion sciFLEXARRAYER S3 (Berlin, Germany) on streptavidin-coated glass slides^{21 (link)}. Recombinant HA was premixed with mouse anti-streptag-Alexa647 and goat-anti mouse-Alexa647 antibodies in a molar ratio of 4:2:1 in PBS-T (50 µl) for 15 min on ice. Sub-arrays (6 × 27 spots) were incubated with the premixed HA for 90 min in a humidified chamber before washing of the whole slide in 4 successive steps with PBS-T, PBS and deionized water with 5-min soak times. Arrays were dried by centrifugation and fluorescence was scanned using an Innopsys InnoScan 710 microarray scanner (Carbonne, France). The data were processed with GenePix Pro 7 (Molecular Devices, San Jose, CA) and analyzed with an in-house developed Excel macro using Excel 2016⁷² . The highest and lowest replicates were removed, and the mean and standard deviation were calculated (n = 4). Data were plotted using GraphPad Prism 7.0 (San Diego, CA).

Glycan arrays were performed as a service by Z Biotech. The array was blocked for 30 min using glycan array blocking buffer (Z Biotech item number 10106). Alexafluor-555-labelled samples were diluted in glycan array assay buffer (Z Biotech item number 10107) to the desired concentrations and then applied directly to the array. The array was covered with an adhesive film and shaken at 80 rpm for 1 h at room temperature. The array was then washed three times with glycan array assay buffer and two times with MilliQ water. The array was read using an Innopsys InnoScan 710 Microarray Scanner with a high-power laser at 1× photomultiplier gain. Proprietary software was used to detect each spot on the array and calculate the relative fluorescence unit (RFU) intensity for each spot. Background RFU was subtracted from each spot’s RFU value. The median of four repeat spots was determined for each glycan.

Purified, soluble trimeric HA was precomplexed with an anti-Strep-tag mouse antibody and Alexa 647-linked anti-mouse IgG (4:2:1 molar ratio) prior to incubation for 15 min on ice in 100 μL phosphate-buffered saline (PBS)–Tween (PBS-T) and incubated on the array surface in a humidified chamber for 90 min. Slides were subsequently washed by successive rinses with PBS-T, PBS, and deionized H₂O. Washed arrays were dried by centrifugation and immediately scanned. The slides were scanned using an Innopsys Innoscan 710 microarray scanner at the appropriate excitation wavelength. To ensure that all signals were in the linear range of the scanner’s detector and to avoid any saturation of the signals various gains and photomultiplier tubes (PMT) values were employed. Images were analyzed with Mapix software (version 8.1.0 Innopsys) and processed with our home written Excel macro. The average fluorescence intensity and SD were measured for each compound after exclusion of the highest and lowest intensities from the spot replicates (n = 4).