Servizi disponibili

MEA assay

MEA arrays measure the electrical activity of large cellular networks in real time without labelling.

• 3BRAIN AG BioCAM DupleX: MEA array system for in vitro functional imaging, compatible with MEA chips with 4096 electrodes. The system works with adherent cells, tissue and organoids slices and allows for recording extracellular voltage oscillations.

Ion Imaging assay

Using a fluorescent ion sensor, it is possible to analyze the electrical activity of a cell by measuring the intracellular ion oscillations using a conventional microscope. This service will be performed using microscopes of the IU1 Imaging.

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The Structural Proteomics unit will perform integrative structural modelling combining medium\low resolution (from 7 to 30 Å) cryo-EM densities and crosslinking MS data acquired at our Facility. In case of very large systems, negative stain data may also be used. This does not refer to model building in cryo-EM densities, but to calculations of localization of subunits or of areas not observed in EM maps. This task can be performed on data from services SB-IU4-A or SB-IU4-B only. Example software: DisVis, integrative modelling platform (IMP), AlphaLink\AlphaLink2. This service will be performed by Facility Staff, but training will be available if requested, provided basic bioinformatics skills of the User (bash terminal, python).

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This service provides high-resolution cryo-TEM data collection of vitrified specimens. According to instrument availability and experimental needs, data collection will be carried out either on 200kV or 300kV microscope systems. A maximum of 1 specimen can be imaged per unit of service. To ensure efficient usage of high-end microscope time, a maximum of 4 grids can be loaded per unit of service. The User can provide cryo-TEM grids either unmounted or mounted on a Thermo Scientific cartridge. In case of an unmounted grid, clipping of the specimen in Thermo Scientific cartridges will be performed by Facility Staff. In case of User-provided already-clipped grids, these will be inspected by Facility Staff prior to acceptance. Imaging at 200 kV will be performed on a Thermo Scientific Glacios while imaging at 300 kV will be performed on a Thermo Scientific Titan Krios G4, both equipped with a Falcon 4i direct electron detector and a Selectris X energy filter. Imaging conditions (i.e., dose, pixel size, magnification, etc.), if requested by the User, must be compatible with the Facility best practices. Microscope access is granted for a maximum of 24 continuous hours per unit of service, including all steps from clipping to loading and TEM alignments and according to Facility Staff availability. For single-particle acquisition, User might opt for beam-image shift assisted data collection (~ 450 – 600 movies\hour) or stage movement for each hole (~ 100 – 250 movies\hour). This service will only be performed by Facility Staff.

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Full-service sorting of rare populations from heterogeneous samples, cell cloning (single cell deposition into multi-well plates), particle enrichment, and high purity bulk sorts.

• High-recovery and indexed single-cell sorting for sequencing.
• Cell sorting of many cell types including immune cell and hematopoietic stem cell subsets, mesenchymal stem cells, viable cytokine producing cells and general cell sorting approaches for cell lines and transfected cells.

Sorter technical features:

• BD FACSDiscover S8
  This instrument is the latest technological advance, representing a breakthrough solution to combine capabilities of cell separation with multiparametric spectral parameter-based analysis and imaging.  Image based cell selection combined with fluorescently labelled tags for determination of tag local. Sorting of up to 6 populations simultaneously with recovery in numerous devices including 384 well microplates. The sorter is equipped with 5 lasers (349 nm, 405 nm, 488 nm, 561 nm, and 637 nm).
• MoFlo Astrios EQ
  The sorter features dual forward scatter for enhanced detection of small particles such as MV’s and capable of very high-speed bulk sorting. Sorting of up to 6 populations simultaneously with recovery in numerous devices including 1536 well microplates. The sorter is equipped with 6 lasers (355 nm, 405 nm, 488 nm, 561 nm, 592 nm, and 640 nm).

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Analysis of cell populations in suspension using conventional, spectral and imaging analyzers.

Analyzer technical features:

• Cytek Aurora Spectral Analyzer
  The analyzer is designed for high-complexity applications, enabling the use of a wide array of new fluorochrome combinations and capable of resolving challenging dye combinations as well as extracting autofluorescence to enhance resolution of dim markers.  The analyzer is equipped with 5 lasers (355 nm, 405 nm, 488 nm, 561 nm, and 640 nm), spectral detection of 64 channels and is equipped with automated sample loader.
• ImageStream Mark II Imaging Flow Cytometer
  This instrument combines the speed, sensitivity, and phenotyping capabilities of flow cytometry with the detailed imagery and functional insights of microscopy. The analyzer is equipped with 6 lasers (375 nm, 405 nm, 488 nm, 561 nm, 592 nm, and 642 nm) and a 785 nm side scatter laser, a 12-channel spectral detector, and multi-channel brightfield.
• CytoFLEX LX Flow Cytometer
  High speed and multi-parametric analysis for immunophenotyping and micro-vesicle analysis with 6 lasers and 21 fluorescence APD detectors. Laser wavelengths include 355, 405, 488, 561, 640, 808 nm. Sample acquisition may be performed in single tubes or in 96-well plates (flat, V or U bottom). Deep-Well plates may also be used for samples requiring lager volumes. In addition to the standard Side Scatter parameter on the 488 nm laser line, an added side-scatter parameter is configured for the 405 nm laser line.

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This service allows exploring the potential of single molecule techniques. It is dedicated to Users with no prior experience with dynamic single molecule (DSM) approaches who would like to obtain ‘proof of concept’ data necessary to plan future experiments. The unit is equipped with two state-of-the-art instruments: (1) c-trap and (2) c-trap edge. The c-trap relies on confocal scanning, and it is predominantly used for imaging in solution. C-trap edge has an option to switch between wide field fluorescence and TIRF. In addition, c-trap edge allows for label free imaging based on IRM, which makes this instrument ideal for imaging close to the surface. The Unit can assist in a very broad spectrum of experimental approaches.

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This service is dedicated to projects where specific single molecule assays have been already established or, alternatively, should be combined with ‘assay development’ service as a simple ‘package’. Examples of experiments currently performed routinely by the unit: (i) detecting changes to DNA structure upon protein binding e.g., melting, looping, compaction; (ii) characterizing protein behaviour upon binding to DNA (diffusion, direct motion, interactions with other proteins). This service can be performed by the Facility Staff or by a trained User. The training can be provided by the unit as part of this service. Full training typically requires 3-5 days. The User can spend up to 10 days at the Facility. The work can be split into two visits. Original data will be handed to the User as .h5 files. The unit will assist the User in accessing the files using Pylake. In case of kymographs, the Facillty Staff can provide a short training on existing tools such as Lakeview.

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Development and execution of custom gene editing projects.

This service is designed to offer expertise and technical support for the implementation of genome engineering projects that demand customized approaches not presently covered by off-the-shelf knock-out and knock-in editing services.

The proposal should provide a thorough explanation of the rational, goals, and expected outcomes to facilitate a comprehensive evaluation.

Tailored gene editing projects involve completing sequential checkpoints before moving on to the implementation of the proposed experiment (Phase 3). Throughout these phases, significant interaction with the applicant is expected.

• Phase 1 (Strategy design): A tailored strategy will be designed following preliminary meetings and discussed with the applicant before proceeding to the next phase.
• Phase 2 (Strategy validation): The designed approach will undergo testing in the relevant cell line to assess its effectiveness (efficiency, locus accessibility, …).
• Phase 3 (Target editing generation): Upon successful completion of Phase 2, the validated approach will be applied to generate the desired cell line.

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This service provides cryo-Fluorescence Microscopy imaging including sample preparation by plunge-freezing and grids clipping. A maximum of 1 specimen can be processed per unit of service, maximum 4 samples (i.e. replicates) can be prepared per specimen. Glow discharging will be performed by either a Pelco EasyGlow, a Quorum GloQube, or Solarus II plasma cleaner device. Plunge-freezing will be performed on a Thermo Scientific Vitrobot Mk IV or a Leica EM GP2. Applicants may provide TEM grid supports of preference, otherwise the Facility Staff will use the available TEM grid supports. Widefield imaging will be performed on a Leica Thunder cryo-CLEM system. Confocal imaging will be performed on a Leica Stellaris 5 cryo-CLEM system equipped with white light laser. Both microscopes are equipped with a 50x \ 0.9 NA lens. This service is provided for a maximum of 8 continuous hours per unit of service, including plunging, clipping, and imaging. In case of User-provided already-clipped grids (in Thermo Scientific cartridges), these will be inspected by Facility Staff prior to acceptance. This service will only be performed by Facility Staff.

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This service provides sample preparation by plunge-freezing, grid clipping for autoloader system and cryo-TEM imaging at 200 kV. A maximum of 1 specimen and 8 grids can be prepared and processed per unit of service. An optional “polishing” SEC step can be performed on thawed material by the Biophysics Unit, as and if specified in the request for this service. Glow discharging will be performed by either a Pelco EasyGlow, a Quorum GloQube, or Solarus II plasma cleaner device. Plunge-freezing will be performed on a Thermo Scientific Vitrobot Mk IV or a Leica EM GP2. Applicants may provide TEM grid supports of preference, otherwise Facility Staff will use holey TEM grid supports available. Imaging will be provided for a maximum of 8 continuous hours per unit of service. Cryo-TEM imaging will be performed only in EF-TEM mode at 200 kV on a Thermo Scientific Glacios equipped with a Falcon 4i direct electron detector and Selectris X energy filter. Imaging conditions requested by the User, if provided, must be compatible with Facility practices. In case of grids showing optimal particle distribution and ice quality, this service can be extended for overnight data collection, if compatible with Facility Staff working hours. The User can also provide already-made cryo-TEM grids either unmounted or mounted on a Thermo Scientific cartridge. In case of an unmounted grid, clipping of the specimen in Thermo Scientific cartridges will be performed by Facility Staff. In case of User-provided already-clipped grids, these will be inspected by Facility Staff prior to acceptance. This service will only be performed by Facility Staff.

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