[Translate to English:] Nos plateformes et nos plateaux techniques

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[Translate to English:] State-of-the-art equipments

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The « Plateau d’ImmunoPhénotypage Métabolique » is a structure, within EGID and UMR 1011, dedicated to the study of immune and endocrine cells from immune and metabolic organs (liver, adipose tissues, muscles) in cardio-metabolic pathologies and their vascular complications (obesity, diabetes, atherosclerosis, hepatic steatosis…).

In this context, we have a gathered a scientific expertise in both immunology and metabolism to carry integrated analyses on human samples or preclinical and experimental models using  both metabolic investigation tools (special diets, glucidic and lipid metabolism, as well as flow and mass cytometry

 

Three instruments are available for cell analysis and sorting:

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-         - A BD LSRFORTESSA X-20 (Becton Dickinson®1), last generation of high-performance multi-color analyzers, which allows simultaneous acquisition of 18 fluorescence and 2 structural parameters.

-         - A BD INFLUX (Becton Dickinson®2), high-performance « jet in air » cell sorter allowing simultaneous acquisition of 21 fluorescence and 2 structural parameters. Very flexible and adaptable to users specific constrains, unique in the North of France, this instrument is one of the few with 6-ways sorting, (extremely useful for optimal use of difficult to obtain samples).

-         - A mass cytometer CyTOF 2 (Fluidigm®1). This unique technology allows simultaneous analysis of more than 40 parameters (upon antibody labelling with stable metal isotopes) at the scale of the individual cell thanks to coupling cytometry to mass spectrometry.

-        -  An analysis station equipped with softwares dedicated to processing of classical as well as multi-parametric cytometry data (FlowJo®, FACSDiva®, Cytobank®, Cytofkit on R).

[Translate to English:] Steering committee

[Translate to English:] Fonction Name Phone Mail
Director Bart Staels 33 (0) 20 87 73 88 bart.staels@pasteur-lille.fr
EGID coordinator Dominique Pacot 33 (0) 3 20 97 42 51 dominique.pacot@univ-lille2.fr
Scientific officer David Dombrowicz 33 (0) 20 87 79 67 david.dombrowicz@pasteur-lille.fr
Technical officer Olivier Molendi-Coste 33 (0) 3 74 00 80 84 olivier.molendi-coste@inserm.fr
Research engineers Laurent Pineau & Olivier Molendi-Coste 33 (0) 3 74 00 80 84 laurent.pineau@inserm.fr / olivier.molendi-coste@inserm.fr

[Translate to English:] Our facilities

[Translate to English:] BD LSRFORTESSA X-20 analyzer

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(Becton Dickinson®1)

 

From the last generation of multi-colors high-performance analyzers, it is equipped with 5 lasers, allowing analysis of 2 physical (size/FCS and structure/SSC) and 18 fluorescence parameters (optic path FORTESSA X20).

Electronic configuration permits acquisition of millions of events at a maximal speed of 20 000 evts/second.

Conception of the systems for acquisition of size/structure signals (PMT on FSC) as well as for sample line and fluidics allows analysis of cellular events ranging from a very small size (0.1µm diameter, such as bacteria) to 40-50 µm diameter.

Technical specifications of the FORTESSA X-20 are accessible here.

User-friendly, access to FORTESSA X-20 is validated by the plateau’s technical staff after an individual training of 3 hours. For booking, please click here.

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[Translate to English:] BD INFLUX cell sorter

[Translate to English:] (Becton Dickinson®)

This « jet in air » cell sorter is highly modular, due to manual control, allowing to adapt its configuration to the specific needs and constrains of users. Moreover, informatics system configuration in digital data offers a better control of acquisition quality and treatment of light signals acquired.

This multi-color, high-performance and high-throughput (20 000 evts/second) instrument is equipped with 5 lasers, allowing simultaneous acquisition of 2 structural (size/FSC and structure/SSC) and 21 fluorescence parameters (INFLUX optic path). The optic configuration of this INFLUX being very close to that of the FORTESSA X-20, it is possible to set up a staining strategy for sorting on the analyzer and to transfer it as such for  the cell sorter.

The « jet in air » technology better preserves cellular integrity when compared to a classical sorter, notably due to reduced hydrostatic pressure, but with an equivalent debit and detection sensitivity.

Unique in the North of France, this instrument is one of the few to propose a 6-ways sorting, ensuring collection of 6 distinct populations of interest, as defined by the staining, from a single sample. It is also possible to sort in 96 wells-plates in “single cell” configuration (cloning). In addition, temperature of the sorting receptacle can be set from 4 to 37°C for better preservation of the sample.

Conception of the “size” acquisition system (PMT on FSC) allows processing of cell entities of very small size, from 0.1 µm diameter (bacteria). Furthermore, the design of the fluidic system (both sample line and nozzle) allows sorting very large cells, up to 100 µm diameter, where classical sorters are limited to 50-60 µm.

Technical specifications of the INFLUX are accessible here.

Of complex use, acquisitions and sorts on the influx are realized by the technical staff of the plateau after 1) a meeting for validation of both the panel and the gating strategy, and 2) experimental validation of the stainings on an analyzer. Click here to contact the “sort-engineers”

[Translate to English:] CyTOF II mass cytometer

[Translate to English:] (Fluidigm®)

Mass cytometry represents a true innovation at the frontier between cytometry and mass spectrometry.

In mass cytometry, antibodies and probes used for identification of target entities are conjugated tostable (non-radioactive) highly purified metal isotopes, which each possess a precise unique relative atomic mass (Dalton, Da). Antibody-lalelled cells are injected in a system comparable to a cytometer (the “Cy” of CyTOF) which collects, drives and sprays the single cells into single drops, which are then ionized with a plasma torch as ion clouds, injected one by one in a mass spectrometer. “Time of flight” (the “TOF” of CyTOF) identifies each ionized metal isotope ion present in the cloud as it is proportional to its relative atomic mass. Intensity of the signal is, for each atomic mass, proportional to the abundance of ions, and thus to the intensity of the specific staining.

Width of the detection spectrum of the TOF from CyTOF II  (from 89 to 209 Da) allows simultaneous analysis of more than 40 parameters in practice, and up to 120 in theory, and this on millions of single cells. Schéma de fonctionnement

Targets can be surface markers, cytokines, transcription factors, mRNA, metabolites, as far as the probes/antibodies can be conjugated to heavy metals. As of today, around 40 markers can be analyzed routinely at the same time in a sample. Illustration de données obtenues

This system of detection, notably its stability in identification of isotopes by the TOF (unique mass) as well as the absence of interferences between isotopes avoids classical compensations between fluorochromes necessary in flux cytometry. Moreover, as the heavy metals used are extremely rare in the living reign (except specific cases), there is almost no background signal akin to autofluorescence.

A dedicated online free access tool has been developed by Fluidigm® in order to help for elaboration and optimization of the panels (photo panel designer). This software allows to evaluate “target expression level/isotope detection intensity” for each marker so as to optimize detection sensitivity in a multi-parameter context.

Panels are validated at the theoretical level with the plateau engineers, and the tests/optimization are performed prior to the full experiment

Mass cytometry technology does not allow for high output such as in flux cytometry. It takes about one hour for acquisition of 1 million cells.

Of omplex use, acquisitions with the CyTOF II are realized by the technical staff of the plateau. Click here to contact the “isotopic-engineers”

[Translate to English:] Analysis Station

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A powerful computer (i7, 16 Go RAM) can be booked to proceed to cytometry analysis. It is equipied with the following softwares:

FlowJo (TreeStar®)

FACS Diva (Becton-Dickinson®)

R and Cytofkit (R foundation and « Chen et al. 2016 »)


For mass cytometry data, which usually contain a high number of parameters, it is necessary to use automated algorhythms for dimension reduction (illustration), clusterization (illustration) and automatic identification of populations (illustration). To this end, the analysis station is implemented with the « cytofkit » R package. The research engineers also have a private license for access to the online analysis software CytoBank®, they can thus help you for the analysis of your data with this software. It also allows formatting and illustration of cytometry data (from flux and mass).

Acces to the analysis station booking here

It is possible to get assisted by the research engineers of the plateau for softwares handling, elaboration of analysis strategies…

To contact the engineers, please click here.

[Translate to English:] Pricing

[Translate to English:] In edition. Please, contact us for more details.

[Translate to English:] Reservations

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For accessing the online reservation web-page, one needs access validation by the technical staff who will provide an individual identifier and password. Then, follow the link below:

FORTESSA : please click here

Poste d’analyse : please click here

[Translate to English:] Partners and supporters of the Plateau

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The technical plateau was supported by FEDER (european) and "Laboratoire d'excellence EGID" (ANR-10-LABX-46) fundings.

The research Unit hosting the plateau (U1011) is supoprted by "INSERM", "Université Lille 2" and "Institut Pasteur de Lille".

[Translate to English:] Useful links

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Spectra viewers for excitation and émission spectrum of fluorochromes developped by different companies: