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Cytometry

Presentation

Flow cytometry is a technology that allows simultaneous multiparametric analysis of the biological, physical and chemical characteristics of particles in a fluid as it passes through at least one laser line. Cell components can be fluorescently labelled and then excited with a laser to emit light at varying wavelengths.

The fluorescence can be measured to determine various properties of single particles, which are usually cells. Up to thousands of particles per second can be analysed as they pass through the liquid stream.

The Flow Cytometry Facility manages the use and maintenance and set-up of the two pieces of equipment present at Biocruces Bizkaia. The Facility also provides with assistance in sample preparation, data analysis and experimental set-up and design.

Facilities analysis programs

FACSDiva: BD FACSDiva software that allows the acquisition of data in the FortessaX20 LSR and its subsequent analysis of data manually or in batch, diagrams with the possibility of linear, logarithmic and bi-exponential scales and export data (FCS 2.0 and FCS 3.0 file format) and to Offline compensation.

MACSQuantify: Software that works with Windows operating system with all the functions to represent data with image analysis and statistics with the possibility of data analysis during the acquisition of samples, data grouping and generation of a single file.

FlowLogic: FlowLogic: Data analysis program that is able to analyze files generated by any flow cytometer. It offers different analysis platforms that allow standard analyzes with different regions that define subpopulations and their statistics.

Services

Portfolio of services

  • Cell immunophenotyping in eight colors.
  • Cell viability assays.
  • Apoptosis analysis. Annexin V + 7-AAD / IP.
  • Cell cycle analysis.
  • Measurement of mitochondrial function.
  • Expression of fluorescent proteins. Measurement of transfection markers such as GFP, YFP, CFP.
  • Cell proliferation assays.
  • Multiparametric analysis of molecules in liquid solutions. Cytokines bead array.
  • Analysis of physiological cellular properties such as oxidative stress, calcium flux and PH.
  • Determination of the cytotoxic capacity of NK cells.
  • Technical and scientific support on Flow Cytometry.
  • Assistance on methodology and experimental design.
  • Advice on acquisition and analysis of samples.
  • Delivery of training courses for users at a basic level.

Service request

For further information and reservation, contact the facility by the web form.

Infrastructures

Currently, the service has the following infrastructure:

FORTESSA X20 analyzer cytometer

The Becton Dickinson cytometer, model Fortessa X20, is equipped with a blue laser (488nm, 50-mW,), a red laser (633 nm, 100-mW) and a violet laser (405 nm, 50-mW).

It consists of two morphological parameter detectors, (FSC and SSC) and 14 fluorescence detectors, analyzing for each of them the area, height and width of the pulse.

It incorporates an optics based on the reflection of light conducted by fiber optics, developed and patented by BD (octagon and trigon system with PMTs coupled to fiber optics) for minimum signal loss and maximum sensitivity.

It has an automated fluid system BD FACSFlow Supply System that ensures the automated supply of system fluids and stabilizes the fluid pressure in the cytometer.

For the analysis of results, it has a software called FACSDiva.

The system includes a quality module that allows to optimize voltages and to control and monitor the operation of the system.

MACSQuant Cytometer from Miltenyi Biotec

The platform also features a MACSQuant® Flow Cytometer. It is an analyzer flow cytometer equipped with 3 wavelength excitation lasers as described below:

  • 405nm. Violet Laser (40mW)
  • 488nm. Blue laser (25mW)
  • 635nm. Red laser (20mW)

It has:

  • 2 Photomultipliers for the Violet Laser (450/50 and 525/50), where Fluorochrome signals such as VioBlue ™, Pacific Blue, Pacific Orange, CFP, BFP, DAPI, ALexaFluor405, DyeCycle Violet, BD HOrizon450 can be collected , Cascade Blue, eFluo450 and Hoeschst.
  • 4 Photomultipliers for the Blue Laser (525/50, 584/40, 655-730, 750LP) that allow working with: Fluorescein (FITC), Phycoerythrin (PE), Propidium Iodide (PI), 7-Actinomycin D (7-AAD), PerCP, Alexa Fluor488,, CFSE, PE-Vio770, PE-Cy5.5, PerCPCy5.5 and PE-Cy7, as well as fluorescent proteins such as GFP, EGFP, EYFP.
  • 2 detectors for the Red laser (655-730 and 750LP) that allow additional work with fluorochromes such as APC, APC-Vio770, Alexa Fluor647, eFluor660, APC-Cy7, APC-H7.

In summary, it allows simultaneous analysis of 10 parameters, 8 optical channels for the simultaneous measurement of fluorescent signals, as well as two PMTs for physical parameters proportional to size (FSC) and cell complexity (SSC).

It can also measure the very important time parameter to be able to perform kinetic analysis.

It is equipped with a robotized support for sample racks (96 plates, 24 tube rack), MACS Cooling Racks that allow keeping the samples at a temperature between 4-8 degrees for two-three hours and by a magnetic column complementary that allows cell enrichment prior to acquisition.

The MACS®Quantify Software (MQS) analysis software can be installed on any computer running Microsoft® Windows® operating system. The software allows the storage in a specific format of the cytometer (MQD) and the export of data in 3 different formats: FCS 3.0, FCS 2.0 and FCS 3.1.

Platform members

Rates

pdf Cytometry platform rates. (Updated January 2020)

Contact form

Contact form - Cytometry

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