Department of Oncology

Description

The BD FACSMelody is a highly automized, 9 colour high speed cell sorter. It is a 4-way cell sorter, equipped with 3 lasers (405nm, 488nm and 561nm), a fixed optical alignment, a pressure-driven fluidics system and a fixed 100um nozzle, ideal to sort most types of tissue.
The BD FACSMelody minimizes manual steps and simplifies the workflow with smart automation. It is equipped with a versatile sort collection chamber that allows users to sort into up to 4-way tubes or multiwells (plates: 6-, 24-, 48-, 96- and 384-well plates software controlled single or multiple cell deposition, one-way sorting) or microscope slide (3 x 9 grid). Cooling and heating for sort collection are available and adjustable through software: 4°C, 22°C, 37°C and 42°C. Sample agitation is adjustable through the software to keep the sample constantly suspended. High sensitivity and resolution to detect and sort dim cells and rare cells and resolve adjacent populations such as low-antigen density cells, small particles and DNA cell cycles
Moreover, the software, named BD FACSChorus™, guides the operator through the entire cell sorting process with its intuitive interface, on-screen instructions and tips and simple-to-read reports.

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Description

The BD FACSCelesta flow cytometer offers multiple configurations to meet varied application needs. The BVR configuration includes Blue, Violet, and Red lasers, optimized to get the most out of the legacy fluorochromes, viability dyes, and BD Horizon Brilliant™ dyes. While a blue laser has long been standard in flow cytometry, the violet laser is gaining popularity as more bright violet-excited fluorochromes are introduced and adopted. The red laser, which optimally excites allophycocyanin (APC) and APC-based tandem dyes, allows markers to be spread across more lasers, further reducing the overall compensation needed for a multicolor panel. Multicolor flow cytometry panel design has presented challenges for researchers, such as varying marker expression, varying dye brightness, and significant emission spillover between fluorescence channels. The combination of new bright, narrow-spectrum fluorochromes and sensitive optics results in panels that can readily resolve even dim populations, yet are easy to use and compensate. The system operates with BD FACSDiva™ software, a collection of convenient and easy-to-use tools for flow cytometer and application setup, data acquisition, and data analysis.

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DIGITAL PCR 1

Description

Digital PCR allows an absolute quantification of a DNA or RNA target with superior accuracy and sensitivity. Using this technology, the analytical sample is automatically distributed on the surface of a dedicated chip, where it is compartmentalized into 20.000 microwells, each virtually occupied by a single molecule. By minimizing the negative effects due to competition among targets, especially in the case of rare sequences, minute amounts of nucleic acids can be amplified and quantified by TaqMan chemistry with fluorescent probes (FAM, VIC), similarly to traditional qPCR. The QuantStudio 3D Digital PCR System platform includes three instruments: (1) a QuantStudio 3D Digital PCR Chip Loader for sample dispensation on the chip, (2) a ProFlex 2x Flat PCR System thermal cycler for the simultaneous amplification of up to 24 chips, (3) a QuantStudio 3D Digital PCR Instrument for data acquisition from chips. Digital PCR can be applied to liquid biopsy, for copy number variation and gene expression analysis, allelic discrimination, rare sequence mining and single-cell analysis.

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• E-mail contact: laboratoriogilardi@unito.it

DIGITAL PCR 2

Description

QuantStudio Absolute Q is a digital PCR platform based on a microfluidic array plate technology that enables all the necessary steps for dPCR—compartmentalizing, thermal cycling, and data acquisition—to be conducted on a single instrument. The whole process, similar to a traditional qPCR, lasts approximately 90 minutes. Dedicated distribution channels allow the simultaneous filling of 20.480 microchambers with high sensitivity, precision and accuracy and assuring the analysis of more than 95% of the loaded sample. Each plate can contain from 4 to 16 samples, interrogating up to 4 fluorescent dyes at the same time (FAM, VIC, ABY, CY5), thus enabling multiplex analyses. Like any other dPCR machine, the instrument gives an absolute count of the specific target(s), measured as copies/microliter of reaction. Beside of a dedicated Master Mix, the instrument works with traditional TaqMan probes (with the option to use SYBR-GREEN as well). Digital PCR is suitable for multiple applications, including liquid biopsy, copy number variation and gene expression analysis, allelic discrimination, rare sequence mining and single-cell analysis.

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• E-mail contact: laboratoriogilardi@unito.it

REAL TIME PCR 1

Description

The ABI PRISM® 7900HT Sequence Detection System is a second-generation sequence detection system instrument designed for automated, high-throughput detection of fluorescent PCR-related chemistries. Carrying a 384-well sample block, the instrument is capable of real-time, end-point, and dissociation curve analysis of assays arrayed on multiple formats. With this instrument it is possible to chose between using a TaqMan® probe in a master mix for the detection of a single target nucleic acid sequence, or the SYBR® Green Double-Stranded DNA Binding Dye 1 used for the detection of double-stranded DNA.
The SDS software performs a series of mathematical transformations on the raw data which refers to the spectral data between 500 nm to 660 nm collected by the SDS software during the sequence detection run.
The ABI PRISM 7900HT Sequence Detection System monitors fluorescent signals generated by several dyes FAM™, NED™, ROX™, SYBR®, TAMRA™, TET™, and VIC™. The Applied Biosystems buffer contains an internal passive reference molecule (ROX™), which acts as a normalization factor for fluorescent emissions detected in the samples. Multicomponenting is the term used for distinguishing the contribution each individual dye and background component makes to the fluorescent spectra detected by the 7900HT instrument. During the multicomponent transformation, the SDS software employs several algorithms to separate the composite spectra from the raw spectrum and then to determine the contribution of each dye in the raw data.

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• E-mail contact: laboratoriogilardi@unito.it

REAL TIME PCR 2

Description

The CFX Opus Real-Time PCR is a real-time PCR system with an integrated 96-well sample block and a touch screen display, that provide sensitive detection for precise quantification and target discrimination, with the possibility to perform a multiplex analysis of 5 targets per well.
The instrument has 6 different filters and can detect emission wavelengths from 450 to 730 nm. CFX Opus systems also include a channel dedicated to FRET chemistry.
Can perform PCR quantification with standard curve, Melt curve analysis, gene expression analysis by relative quantity (∆Cq), Allelic discrimination and End-point analysis.
CFX Opus system’s file browser feature allows to create and manage stored data files that could be transferr by different options: Bio-Rad’s BR.io app allows to manage PCR file and data analysis with the possibility to create real-time PCR experiments in the app, download and run them on CFX Opus system, and then analyze the results.
Another option is the use of wireless network (WiFi), Ethernet, and USB, when successfully connected it is possible to send email notifications directly to a specified user account and access shared network folder.

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• E-mail contact: laboratoriogilardi@unito.it

Description

TellDx is a system for the isolation of circulating tumor cells (CTC) from liquid biopsy samples. The TellDx platform consists of the CTC Isolator II, which is used in conjunction with a disposable CTC iChip. The CTC Isolator II is a standalone, bench-top instrument intended to provide biophysical support, mechanical alignment, and fluidic supply to the microfluidic Chip. Within the Chip, the blood sample, preincubated with magnetic beads conjugated with antibodies recognizing white blood cells, is first subjected to hydrodynamic cell sorting for size-based separation of red blood cells and platelets. Then, inertial focusing generates a single central stream of cells and magnetopheresis deflects beads-tagged white blood cells, resulting in viable CTC enrichment and recovery. This negative depletion strategy is completely agnostic regarding expression of CTC-specific markers, this allowing preservation of the whole population and its heterogeneity. Once collected, CTC can be stained and enumerated, used for minimal residual disease monitoring, exploited for molecular characterization and single-cell analyses, or put in culture for functional studies.

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Description

Next-generation sequencing (NGS), known as "high throughput" and/or "massively-parallel sequencing" allows to investigate a large number of applications, including targeted and de novo nucleotide sequence of deoxyribonucleic acids (DNA) and ribonucleic acids (RNA), quickly and cost-effectively compared with orthogonal methods such as Sanger or Pyro-Sequencing technology. This approach has had a great impact on several fields of molecular research for basic and applied science. The introduction of these technologies within international and national laboratories supported a great scientific innovation resulted from a “multi-target” study approach. NGS technology is able to investigated hundreds of genes simultaneously in several samples for each sequencing run, testing single or multiple nucleotide variants (SNVs/MNV), insertion and/ or deletion (INDEL), aberrant RNA transcripts and copy number variations (CNV) with 99% of limit of detection (LOD) performance, specificity, sensibility, accuracy, repeatability and 95-99% of reproducibility for each test. NGS technology has rapidly evolved over the past 15 years and new methods are continually being commercialized. In our translational research laboratory, NGS analysis, were performed since 2019 by Ion GeneStudio™ S5 Prime Sequencers with custom or commercial broad panel necessary to elaborate a targeted sequencing approach. GeneStudio S5 belongs to Thermo-Fisher brand and several versions of their platform are available, in particular this version supported a broad range of high-throughput sequencing for clinical research. Ion Torrent™ technology directly translates nucleotide sequence (A, C, G, T) into digital information on a semiconductor chip. A “proton chemistry” that applied to semiconductor technology provides fast, accurate and scalable sequencing results. The instrument's advanced technology enables it to provide high-quality sequencing data, even for complex and difficult samples such as samples obtained from histological or cytological preparations by samples formalin-fixed and paraffine-embedded (FFPE). This sequencer is equipped with Torrent Suite™ Software 5.18 (MAN0026467 Rev. D.0) for real-time data analysis and Ion Chef™ System (MAN0017275 Revision D.0) for automated genomic library preparation and chip loading

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• E-mail contact: laboratoriogilardi@unito.it