Scientific facilities

Flow cytometry

Facility Head

Berta Raposo

Scientific Lead

María Luisa Toribio

Contact details

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91 196 44 99

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citometria (at) cbm.csic.es

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At the Flow cytometry facility of the CBM, our staff provides scientific-technical support on flow cytometry, develops and optimizes new applications and performs cell separations.

Flow cytometry is an analytical tool that allows discrimination of particles of different size and colour. It is an analytical and/or preparative technique based on the simultaneous analysis of several parameters of optical nature, quickly and on a large number of individualized particles. This analysis is performed on each particle based on light scattering and fluorescence emission of particles illuminated by a light source.

The parameters that can be analyzed by flow cytometry are: i) those related to the physical characteristics of the particle: size and complexity and ii) the different fluorescence associated with the particle.

The analysis is performed at rates of thousands of cells/second, which allows obtaining data of high statistical reliability, as well as identifying populations represented at low frequency within the overall population.

Another important feature of flow cytometry is that the analysis is performed in a particle suspension, so the particles or cells under analysis must be in suspension and in the form of a single particle (cell).

Finally, it is important to note that there is a restriction on the size of the particles under analysis, which must be between 0.5 and 100 μm.

Vacío

Conventional cytometry

FACSCalibur (Becton Dickinson)
Technical features:
Two laser lines: blue (488nm) and red (633nm).
4 independent fluorescence detectors:
3 detectors for the 488nm laser (blue): 530/30, 585/42, 670LP.
1 detectors for the 633nm (red) laser:660/20.

Software: Cell Quest-Pro

FACSCanto A (Becton Dickinson)
Technical features:
Two laser lines: blue (488nm) and red (633nm) with 6 independent fluorescence detectors:
4 detectors for the 488nm laser (blue): 530/30, 585/42, 695/40, 780/60
2 detectors for the 633nm (red) laser:660/20, 780/60

Software: DiVA 8.0.1

FACSCanto II High Throughput Sampler Option (Becton Dickinson)
Technical features:
Equipped with a High Throughput Sampler Option (HTS) system for sample acquisition directly from multiwell plate.
Three laser lines: blue (488nm), red (633nm) and violet (405nm)
8 independent fluorescence detectors:

4 detectors for the 488nm laser (blue): 530/30, 585/42, 695/40, 780/60
2 detectors for the 633nm (red) laser:660/20, 780/60
2 detectors for the 405 nm (violet) laser: 450/50, 575/26

Software: DiVA 8.0.1

FACSCanto II High (tubes only) (Becton Dickinson)
Technical features:
Equipped with a High Throughput Sampler Option (HTS) system for sample acquisition directly from multiwell plate.
Three laser lines: blue (488nm), red (633nm) and violet (405nm)
8 independent fluorescence detectors:
4 detectors for the 488nm laser (blue): 530/30, 585/42, 695/40, 780/60
2 detectors for the 633nm (red) laser: 660/20, 780/60
2 detectors for the 405 nm (violet) laser: 450/50, 605/10

Software: DiVA 8.0.1

Spectral cytometry

AURORA 4L (Cytek)
Optical Configuration: 4L (16V-14B-10YG-8R)
4 laser lines and 48 detectors: 16 detectors for the violet laser (405nm), 14 detectors for the blue (488nm), 10 detectors for the yellow green (561nm) and 8 detectors for the red (633nm).
Technical Characteristics: This equipment uses full spectrum technology to provide unprecedented flexibility to users, enabling them to use of a wide range of new fluorochrome combinations without reconfiguring the system for each application. This technology provides high-resolution data at the single-cell level to resolve the most challenging cell populations, such as those with high autofluorescence or low levels of marker expression.

AURORA 5L (Cytek)
Optical Configuration: 5L (16UV-16V-14B-10YG-8R)
4 laser lines and 64 detectors: 16 detectors for the ultraviolet laser (355nm),16 detectors for the violet laser (405nm), 14 detectors for the blue (488nm), 10 detectors for the yellow green (561nm) and 8 detectors for the red (633nm).
Technical Characteristics: This equipment uses full spectrum technology to provide unprecedented flexibility to users, enabling them to use of a wide range of new fluorochrome combinations without reconfiguring the system for each application. This technology provides high-resolution data at the single-cell level to resolve the most challenging cell populations, such as those with high autofluorescence or low levels of marker expression.

Cell sorters

FACSAria Fusion (Becton Dickinson)
The BD FACSAria Fusion is an easy-to-use cell separator that allows the separation of a large number of samples of different origins. It is installed in a Class II Type A2 biosafety cabinet equipped with an integrated aerosol containment system that operates independently. Some technical features are detailed below:
Equipped with four excitation lasers 405 ,488, 561and 640 nm. Eighteen detectors (16 fluorescence + 2 scattering) of which 5 are for the 405nm laser (450/40, 525/50, 610/20, 660/20, 710/50, 780/60), 2 for the 488nm laser: 530/30, 695/20, 4 for the 561nm laser: 610/20, 670/14, 710/50, 780/60 and 3 for the 640nm laser: 670/30, 730/45, 780/60. It is also equipped with a temperature control system and sample agitation.

Work stations and software

The service provides users with three analysis stations (Hewlett-Packard) equipped with the following software packages:

– Station 1: FlowJo v10 and 7.6.5, Spectroflo3.3.1 (4 or 5 L configuration)

– Station 2: FlowJo v10, Spectroflo 3.3. 1 (4 L configuration)

– Station 3: FlowJo v10, Spectroflo 3.3.1 (5 L configuration), Gen5 and DIVA 9, as well as an online license of OMIQ software (a modern, cloud-based flow cytometry analysis platform bridging machine learning and analytical pipelines with classical analysis).

Other equipment

Cytation (Agilent)
Technical Features: The BioTek Cytation 1 multimode cell imager combines fluorescence and high contrast brightfield imaging with conventional multimode detection.
This microscope is equipped with a 4X objective, and 3 fluorescence lines:
– DAPI (EX 377/50 nm, EM 447/60 nm); LED 365 nm.
– GFP (EX 469/35 nm, EM 525/39 nm); LED 465 nm
– RFP (EX 531/40 nm, EM 593/40 nm); LED 523 nm
Gen5 software simplifies image capture and plate reading.

eFlux Analyzer XF96 (Agilent)
Technical Features: The Extracellular Flux Analyzer XF96 (Agilent-Seahorse) allows the characterization of the energy metabolism of cultured cells in a non-invasive way. The instrument analyzes oxygen consumption (OCR) and extracellular acidification (ECAR, an indirect measure of lactate production) in 96-well plates. Our service provides access to the equipment as well as basic assistance in both experiment design and data analysis.

NanoSight NS300 (Malvern Panalytical)
Technical Features: The NanoSight NS300 uses nanoparticle tracking analysis (NTA) technology, which takes advantage of the properties of light scattering and Brownian motion to obtain measurements of the concentration and size distribution of particles in liquid suspension.
– Size range: 10 – 1000 nm
– Concentration range: 106 – 109 particles/ml
– Temperature control range: 5°C below ambient temperature to 50°C
– Minimum sample volume: 250 µl
– Chamber: SCMOS
– Focusing: Computer-controlled motorized focusing

Applications:
– Molecular and particle size distribution analysis.