Platforms

Flow cytometry platform

Our Research Unit has massively invested to setup a flow cytometry facility, which comprises :


BD ACCURI C6

2 lasers, 4 colors
C6 HELP PAGELink to booking.

C6


BioPlex Luminex (BioRad)

2 lasers, 3 colors, High-Throughput System for multiplex cytometric bead arrays
Link to booking.

luminex


LSRII (BD Biosciences)

3 lasers, 17 colors with a High-Throughput System
LSRII HELP PAGELink to booking.

LSRII


FACSARIA III cell sorter (BD Biosciences)

5 lasers, 15 colors, 4 way sorting on plates, slides or tubes (acquisition for the Bichat site with grants obtained from the CODDIM, INSERM and the Université Denis Diderot).
ARIAIII HELP PAGELink to booking.

ariaIII

These devices allow to perform analytical flow cytometry on vascular or hematopoeitic cells or microparticles. We have an extended expertise for multiplexed measurements of analytes with cytometric bead arrays (CBA, Luminex, eBiosciences and customized beads). The new cell sorter provides us with phenotypically pure cell preparations for further cell cultures or molecular biology.
Currently, we do not have a dedicated core lab engineer and our strategy is to promote internal trainings of our users (2 training sessions per year are organized by A Nicoletti). We now have a consistent number of skilled users that can in turn train the beginners.
Given the high level of equipment, besides its update, it is not planned to expand this core lab in the near future with additional devices.

Animal house

Our Research Unit has its own animal facility and experimentation rooms (Table below for description). We host and breed a high number of mouse strains, rats and rabbits. We are at the beginning of upgrading the animal facility. Mice are now bred in individually ventilated caging systems (Allentown) that has allowed to both increase the density of housing (150 -> 305 cages) and to warrant an improved health status (we have acquired an animal transfer station and a bedding disposal station). We have implemented a computerized management system of our breedings that will be extended to all animals housed in our core facility.

Pasted Graphic 1

In addition, our animal core facility comprises 3 labs equipped with surgical microscopes and anesthesia stands. One room is dedicated to experimental X-ray experiments.
During the next mandate, we wish to deeply re-organize our animal department to increase its surface, to consolidate all breedings and experimentation labs in the same protected area. We are currently negotiating with the Hospital administration and Inserm to obtain the required space.

xCelligence system

Informations xCELLigence 2012 -4

L’appareil de mesure d’impédance cellulaire xCELLigence est installé dans la salle de culture cellulaire 4ème étage gauche. Celui-ci est dans l’étuve haute à côté de la chambre d’hypoxie.

Il est équipé de 3 modules qui peuvent fonctionner indépendamment.

Son utilisation est soumise à réservation via le Booking system.

Les personnes souhaitant utiliser ce système sont priées de se rapprocher de Jamila.

Afin d’optimiser son utilisation, il est EXPRESSEMENT demandé aux utilisateurs de préciser sur leur plage de réservation :
– Nombre de plaques (1 à 3)
– Utilisation : étuve classique ou hypoxie

Lien vers le site du producteur (plusieurs protocoles sont disponibles pour l’analyse de l’adhérence et de la prolifération cellulaire, la migration cellulaire, des études de signalisation,…).

TECAN

TecanTecan2
Lecteur multi-technologie monochromateur
INFINITE M200 PRO

La lampe Xénon Flash couvre toute la gamme de longueur d’onde de 230 nm à 1000nm (spectres d’absorbance et fluorescence).

L’INFINITE M200 permet de travailler sur des plaques de 6, 12, 24, 48, 96 et 384 puits mais aussi sur des supports tels que lames de verre, boites de Pétri, support « fait maison » etc….

– Le module d’absorbance couvre le domaine UV/VIS de longueur d’ondes de 230 à 1000 nm.
– Le module de fluorescence permet de faire des mesures (Excitation 230-850 nm, Emission 250-850 nm), en Fluorescence « classique » mais également en TRF, FRET, TR-FRET.
– Le module de luminescence permet des mesures en luminescence Flash, Glow, DLR.
– Module pour la technologie Bioluminescence Resonance Energy Transfer (BRET)

L’instrument possède un module de régulation de température, un système d’injection, un module de régulation de gaz CO2 (0-10%) et/ou O2 (0.5 – 21%), une fonction d’agitation, une multi-lecture par puits

L’appareil est installé au 3ème étage pièce du bout à droite.

Son utilisation est soumise à réservation via le Booking system.

Les personnes souhaitant utiliser ce système sont priées de se rapprocher d’Angélique.

Platelet function platform

Our unit has invested in technologies allowing characterizing human and mouse platelet functions.
The “platelet platform” has many applications including:
• Basic research studies on the physiologic role of platelets in hemostasis and their pathologic role in thrombosis
• Identification of platelet defects in patients
• Clinical studies: diagnostic markers and platelet responsiveness to therapeutic agents

Bioengineery: analysis of the biocompatibility, hemostatic toxicity of polymers and devices; development of new tracers.
The platform includes:
• Platelet quantification and volume: Sysmex XE
Human: Hematology laboratory of the hospital
Mouse: SCIL Vet ABC

hemato

• In vitro bleeding time PFA100 Siemens
• Platelet phenotype: analysis of the characteristics platelet receptors by flow cytomery (see above), immunoblot and immunoprecipitation.
• Platelet activation: Expression of activation markers by flow cytometry; Platelet aggregation: light aggregometry (4 channels aggregometer : APACT 40004, Elitech; Chrono-Log)
• Platelet procoagulant activity: thrombinography on platelet rich plasma consists in measuring the catalytic activity of platelets on the formation of thrombin

pleteletbench
“Platelet” bench Aggregometers and Thrombinography from left to right

• Platelet adhesion in static conditions: colorimetric analysis
• Shear-induced platelet activation: “Couette” devices Hematology laboratory Hospital. The principle is to submit platelets to increasing shear rates in order to reproduce the conditions of a stenosed vessel. Platelet activation is measured as the disappearance of individual platelets.

sipagreg
SIPAGREG: “Couette” device adapted for shear induced platelet aggregation

•  In vitro thrombosis: The laboratory has the possibility to analyze the capacity of platelets to form thrombi in whole blood, platelets being labeled with a fluorescent probe (DiOC6 or rhodamine). Two different kinds of parallel plate flow chambers are available: the classical Maastricht chamber and more recently small volume dispensable chambers from Cellix (Dublin, Ireland) either coated with purified proteins or covered by an endothelial cell monolayer.
• Platelet adhesion is monitored in real time using fluorescence microscopy with a monochromator epifluorescence illumination system (DMRIB Leica) and a DP30W camera (Olympus).

left
right
From top to bottom: “Maastricht” parallel flow chamber, Cellix Vena8 endothelial+TM, Vena8 Fluoro+TM chambers

In vivo thrombosis is studied using the ferric chloride model platelets being labeled in vivo by the injection of rhodamine 6G. Arterioles and venules are visualized with an inverted epifluorescent microscope (×10) (Nikon Eclipse TE2000-U), coupled to Metamorph7.0r1 software (Universal Imaging Corporation). Platelet deposition and thrombus growth in arterioles and venules are monitored in real-time until complete occlusion occurred. The analyzed parameters are the vessel occlusion time and the surface and volume of platelet aggregates.

microscopy
Microscopy platform used to analyze in vitro thrombosis in flow chambers and in vivo thrombosis.

In vivo fibrinolysis. A new and original method to study fibrinolysis in vivo has been developed in which ferric chloride thrombosis is triggered on vessels exposed on a dorsal skinfold chamber. These methods allow studying clot lysis and its pharmacological regulation by intravital microscopy on animals maintained alive.

Proteomics

We have developed different complementary proteomic techniques for the discovery of cardiovascular biomarkers and assessment of interactions. Protein profiling for differential proteomics is performed either by bidimensional electrophoresis (2DE) or by mass spectrometry (more appropriate for detection of peptide and protein fragments). In house available equipments:
– 2DE system including two isoelectrofocusing systems (IEF Cell 12 strips, Biorad Biosystems) allowing simultaneous separation of 12 samples according to their isoelectric point, and a twelve-gel PAGE migration system (Ettan Twelve, GE Healthcare)
– dedicated scanner for gel analysis (brightfield: GS800 densitometer, Biorad, fluorescence: chemi-smart Vilbert-Lourmat)
– SELDI-TOF mass spectrometer 4000 enterprise (surface-enhanced laser desorption ionization time of flight, Biorad Biosystems)

proteomic

Biomarkers of interest are identified by advanced mass spectrometry (MALDI MS-MS, Orbitrap…) in collaboration with the Institut Jacques Monod (IFR02 Claude Bernard). MALDI Imaging is available in collaboration with Pr Bedossa laboratory.
For assessment of interactions, label-free system using surface plasmon resonance (Biacore X100, GE Healthcare) is available (own equipment), with a specialized engineer (O H). Finally, we have a full access to a peptide synthesizer than can be used to produce antibodies against peptides of interest or for testing potential therapeutic peptides in vivo (M Lecouvey, Paris XIII University, co-funding).
For 2DE and SELDI-TOF MS, Olivier Meilhac’s group provides the technical and scientific environment for projects that involve proteomics.

Immunohistochemistry facility

Histology facility is composed of 3 separated areas: embedding and sectioning lab, immunostaining (fluorescence and brightfield) lab equipped with fume hoods for classical histological staining and a room for image capture and analysis. Both tissue sections (for brightfield, fluorescence and electron microscopy observation) and cytological analysis (cells grown on slides or cytospun- Rotina 380 centrifuge) are possible within the Research Unit.

IHC
• Paraffin embedding/sectionning:

• Resin embedding:
Tissue embedding in special resins is required for transmission electron microscopy as well as for analysis of hard samples (stented vessels, calcified tissues). Samples should be cropped using a linear precision saw (Buehler ISOMET 5000) and polished (Buehler METASERV 250 grinder-polisher) before sectioning. Classical microtome equipped with a gem grade diamond knife (5-15 um). Ultra-thin sections are obtained using Reichert-Jung Ultracut-E, for electron miscroscopy analysis (performed at the Institut Claude Bernard- Bichat faculty of medicine)
• Cryosectioning:
Microm HM525 cryotome
• Observation: AxiObserver Zeiss (Booking System) and several fluorescence microscopes

Biobanks

Our Research Unit has established a CV biobank collecting a large range of samples (blood, tissue, cells) from the different forms of human CV diseases, including atherothrombotic and non-atherothrombotic diseases. This biobank allows us to test similarities and differences among these pathological conditions. The specific contribution of the major risk factors and now peri-odontal diseases, are addressed. The samples are accessible to a large panel of internal and external collaborations. Some of these collections are associated with clinical databases.

biobank
The main collections include:

  • Carotid artery diseases (1000 samples), which offer opportunities both to collect plasma and serum before surgery, and diseased tissues during surgery (carotid endarterectomy) and due to their anatomic accessibility, to easily develop specific imaging approaches (carotid database).

These biobanking activities include:
Preparation, performance and use of these collections integrate numerous translational innovative methods including informatic management, preparation of DNA, RNA, and chromatin, proteome secretion and extraction, at both tissue and cell levels, permitting downstream genetic and epigenetic analyses.
These CV collections are hosted in the CV BRC of the Inserm building. These CV biobanking activities are integrated in the national Biobank network (Biobank Infrastructure), which are partners of EU BBMRI (www.bbmri.eu). The BBMRI French node is harboured by INSERM.

Link to consult the databases

Preclinical imaging platform

The platform equipment for in vivo imaging of small mammals includes ([iso] means iso9001:2000 certification):

imaging

• Non-invasive blood pressure measurement :Tail-cuff device for mice and rat: Phymep and AD Instruments [iso]
• Metabolic cages for mice : Phymep and Marty technology [iso]
• Electrocardiography Surface recording and analysis : Emka IOX 1.8 and ECG-Auto software 1.5.12.36 [iso]
• Biochemical multiparametric automate : Olympus AU400 [iso]
• Hemodynamics : Millar catheter 1.4F, 2F, and 1.2F for mice and rats, and Emka IOX 1.8 software [iso]
• Telemetry ECG recording : Mouse transmitters DSI and Emka IOX 2.2 software and ECG-Auto 2.1.4 [iso]
• Doppler echography :
– Toshiba Power Vision 6000 workstation equipped with an 8-14 MHz linear probe for mice and rats, and a 6-10 MHz sectorial probe for mice, rats and rabbits [iso]
– Vevo 2100, (Visualsonics) [iso] with 9-18 MHz and 22-55 MHz probes and software for myocardial strain analysis (obtained 2010)
• SPECT imaging devices:
– Gamma-camera DST-XL General Electric gamma camera for large animals
– γIMAGER Biospace Lab, with 2 fixed (non rotative) detectors, planar and pinhole collimators, for mice, rats and rabbits
– High resolution gamma-camera coupled with CT: « NanoSPECT/CT Multi-pinholes » (Bioscan) with « Nuclide » and MMP-SPECT software for mice, rats, and rabbits (obtained 2010)
• PET imaging device : « Mosaic HP Lyso PET », (Philips, Bioscan) with SYNTEGRA software for mice, rats and rabbits (obtained 2010)
• Quantitative autoradiography device : Beta IMAGER™ 3374, Biopace Lab, for quantitative autoradiography of tissue sections (50micrometer resolution).
• For in vitro evaluation of radiotracers

• For radiolabelling of tracers with positron emitter Gallium-68

PhysicoChemical platform

In addition to dedicated roos for chemistry (organic and polymer), our Unit provides to its members its own equipment allowing the physico-chemical characterization of polymers and colloids such as :

PhysChem

¬ The determination of polymer properties : fusion & crystallization events, glass transition, chemical functions, composition, absolute molar mass, refractive index, dn/dc values, contact angle, viscosity, etc… can be achieved with Differential Scanning Calorimetry (DSC), Fournier Transform Infra Red (FT-IR), UV and fluorescent spectrometers, Nuclear Magnetic Resonance 400 & 500 MHz (NMR-share equipment P13), MultiAngle Laser Light Scattering (MALLS – collaboration), Refractometer, Contact angle goniometer, Rheometer.
¬ The determination of colloidal properties : second virial coefficient, translational diffusion coefficient, radius of gyration, hydrodynamic radius, polydispersity, surface charge, shape, two-dimensional image, visualization, etc… are achieved with MALLS, Dynamic Light Scattering (DLS), Laser Doppler Velocymetry, Atomic force Microscopy (AFM- share equipment P13), Field-Emission Gun Transmission Electron Microscope (FEG-TEM- share equipment P13), Scanning electron Microscopy (SEM – share equipment P13).

Network Attached Storage (NAS)

We have 2 NAS or Network Attached Storage: one principal and a second backup one.

The purpose of NAS is to store and centralize data storage for local networks. The act of file serving allows for computer data to be stored on the NAS and to be easily accessible from any where within the local network. This allows for computer data to be quickly disseminated amongst its users and to share data between various computers of the lab. This NAS is accessible only from the lab and from the university building of Bichat. As such, it is not accessible from the hospital but we may ask for specific needs. It is possible to connect from outside (from home for instance) through VPN secured tunnel connections. This requires a specific registration through the INSERM IT service (ask Tony for the exact procedure).
Our main NAS

NAS

This is the RS2212+ from Synology (www.synology.com).
Synology® RackStation RS2212+/RS2212RP+ offers a high-performance, scalable, and full-featured network attached storage solution that meets the needs of business that requires an efficient way to centralize data protection, simplify data management, and rapidly scale storage capacity with minimal time spent on setup and management.
For the time being, the capacity of our two NAS is 65 Teraoctets (65 000 Gb). It can support that one of the hard drives fail without compromising the integrity of the data. The main NAS is backup every night to a secondary NAS.

Important notes

• While the NAS is secured with redundant hard-drives, we cannot be certain that it will never fail, be damaged or stolen. Backup your key data on another support.

• The capacity is large but this should not prevent you from cleaning your respective working spaces.

• The password to access the NAS has been provided to your Team leader that will provide it to you upon request.

• Team-specific storage spaces are shared by all members of the Team. This means that all members from a given team have full access — write | read | erase — to the data of the other members of the team.

• The ‘Commun’ working space is not a garbage storage space. Please keep it clean.

Macroscope 3ème EST

A. MacroFluo Leica 

Capture d’écran 2015-06-08 à 15.11.23

3 objectifs disponibles (1x, 2x, 5x)

Platine chauffante

Polariseur pour objectifs 1x et 2x

4 Filtres fluo :

Y3 ET,k. Excitation BP545/30x, dichroique 570nm LP, émission BP610/75

L5 ET,k. Excitation BP480/40, dichroique 505nm LP, émission BP527/30

A4 ET pour excitation dans l’UV (360/40 nm) Dichroïque 400 DCLP Filtre d’arrêt 470/40 nm

FITC/Rhodamine

Y5 ET,k. Excitation BP620/60,dichroique 660nm LP, émission BP700/75  nm

 

B. Camera Noir et blanc sCMOS Orca Flash 4.0 (Hamamatsu)

C. 1 vibrometre doppler Polytec pour mesure des vitesses

D. 1 laser micropoint

1 Station d’anesthésie Isoflurane

Applications principales:

  • Microscopie intravitale Rat/Souris
  • Observation whole mount

Responsable appareil :

Benoit Ho-Tin-Noé