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Cytometry and Advanced Optical Microscopy Platform

The Cytometry and Advanced Optical Microscopy Platform has the UNE-EN ISO 9001: 2008 obtained in 2012 with the following scope:

  • Advanced optical microscopy, analytical cytometry and cell separation service.
  • Analytical cytometry analysis for investigational drugs.
María Paz López Mato, immunologist(BIR)
mplopez@inbiomed.org - ext 240

In September 2015 Inbiomed has been authorized as a pharmaceutical laboratory for Analysis by Analytical Flow Cytometry for Advanced Therapy Medicinal Products with authorization number 6581E, becoming the first state laboratory holding this authorization.

The Cytometry and Advanced Optical Microscopy Platform enables its users to carry out their experiments in cytometry, cell sorting and confocal optical microscopy with the highest quality standards. It counts on cutting-edge technology and highly trained, specialized staff, which guarantees quality.

Currently the platform performs multi-parameter electrostatic cell sorting processes with different criteria (maximum purity, maximum or intermediate recovery) for different cell types and different eventual applications (cell culture, PCR, Western Blot, etc.).

In addition, the sorting equipment includes a plate-based cell cloning module. It is possible to obtain a sample separated into different final containers: multi-well plates, slides, eppendorf tubes, 12x75 tubes, 15-ml conical tubes, etc.

Furthermore, it is possible to perform a magnetic cell-sorting process in other devices used to this effect, which are also present in the platform.

Analytical cytometry processes are carried out in the platform’s two analysis devices; it is possible to perform multi-parameter cytometry in different applications (cell cycle, apoptosis, intra-cellular and intra-nuclear proteins, cell viability, proliferation, multiplex assays, etc.).

In the area of advanced optical microscopy we acquire digital images from biological samples by means of conventional optical fluorescence microscopy and confocal microscopy, for example: Z-sectioning processes, time lapse short term experiments, emission fingerprints and co-location tests for multiple proteins with different stain types.

In addition, the platform offers a technical support service regarding experimental planning, and provides in-depth support regarding the interpretation of results in a way that is closer to the end user while obtaining results and maintaining data privacy and confidentiality at all times.

FLOW CYTOMETRY AND CELL SORTING

FLOW CYTOMETRY

Flow cytometry is probably one of the most advanced technologies of recent years. The possibility of identifying cell populations efficiently has been of critical importance for the phenotypic and functional characterization of cell populations and their study in processes of differentiation and functionality. Moreover, together with analytical flow cytometry, systems have been developed for the selection, enrichment and purification of target cells that will eventually be used in biomedical applications, which is the purpose of the Inbiomed Foundation.

Flow cytometry allows fast, reliable identification and selection of cell populations based on criteria of light scattering and fluorescence. The presence of multiple laser sources in flow cytometers and sorters -allowing us to use different fluorescence types- offer a vastly improved possibility to identify populations of interest.

The principle of a flow cytometer is that fluorescent light is generated from a light emission source (laser) to intercept a single cell in a predetermined place; this interception will result, on the one hand, in light scattering:

  • Forward Scatter.
  • Side Scatter.

Moreover, if we have stained this cell with antibodies or fluorescent substances capable of absorbing light in the wavelength of the laser being used, the fluorescent molecules will exhibit the property of emitting light in a different wavelength.

All this photon generation will be transformed and collected by an electronic system and processed by a digital system.

In this way, by using one or more lasers we can use from 1 to 6-8-10 or more fluorescent molecules simultaneously, obtaining information on the characteristics of a certain cell.

This basic process of converting light to electrons and to a digital signal is carried out in microseconds; thus, the power of flow cytometry analysis allows the characterization of large numbers of cells in little time.

Basically, a flow cytometer has three main components:

  1. Optics system.
  2. Electronic system.
  3. Fluid system.

In turn, the optics system has two main components:

  1. Excitation optics: this section includes excitation sources (lasers) and a mirror system that conducts this light beam to the flow cell where they intercept the sample.
  2. 2. Collection optics: It is a system of filters and mirrors that direct each of the different wavelengths generated by the laser excitation to their corresponding place on the photomultipliers.s
BD FACSCalibur Fluid System

The fluid system is in charge of sending the sample to the flow cell, where the laser beam-cell interception takes place at the interrogation point.

It consists of:

  1. Sheath fluid container: pressurized isotonic fluid that is sent to the flow cell, where a pressure differential (hydrodynamic focus) is generated between the sheath fluid and the fluid coming from the sample tube. This helps direct the sample to the flow cell and interrogation point.
  2. Waste container: deposit where the sample is sent after it has been analyzed.
  3. Sample injection port: it is where the tube or plate is inserted and from where the sample starts rise once suitable pressure is applied.
  4. Saline filter: 0.22-micron filter that filters the sheath fluid before it reaches the flow cell.

The light scattering signal will be channeled via transmission or reflection mechanisms and then picked up by two types of photo-detectors:

  1. 1. Photodiodes: they capture strong optical signals such as forward scatter (FSC), even though in some cytometers it is possible to substitute a photodiode with a photo-multiplier tube for the FSC.
  2. 2. Photo multipliers: they pick up the side scatter signal. Ideal for weak signals such as side scatter and fluorescence. High sensitivity.
BD FACSCalibur optics/electronics
BD FACSCanto optics/electronics

Then the electronic conversion to analog or digital takes place by means of analog-digital converters that position the cells according to their voltage at a predetermined site in the analysis diagram.

CELL SORTING

In addition to being an analysis cytometer with the ability of providing information on cell properties, the cell sorter is a system that allows the selection and sorting of cells from a heterogeneous mixture.

Basically, the cell suspension goes to the middle of a fluid stream and the cells go through a laser beam where their characteristics are measured. The difference with an analysis cytometer starts here. A vibration mechanism “breaks” the fluid stream into individual droplets, which are then charged positively or negatively by an electrostatic system. After going through deflector plates and according to their charge and/or what we want to collect after sorting, the droplets “are pushed” to different containers.

BD FACSAria III sorting chamber

ADVANCED OPTICAL MICROSCOPY

CONFOCAL MICROSCOPY

Confocal microscopes are instruments that use optical image techniques to increase contrast and/or reconstruct tridimensional images by means of a spatial pinhole (delimiting pinhole collimator) to eliminate unfocused light or lens flashes in specimens that are thicker than the focal plane.

It allows the study of samples with fluorescent marking, making optical sections of them. The sample is excited point by point by a laser light. The emission wavelength of this sample is higher than that of excitation, and it is the latter that when passing through the pinhole allows the detection of a single focal plane.

It makes the tridimensional study of the samples possible, including the interior, and in certain materials it obtains images of the cell surface by reflected light.

Confocal microscopy is also applied in the study of in vivo samples through a time sequence or time-lapse, or for the co-location of different markers in a definite region.

Carl Zeiss Courtesy

FLUORESCENCE MICROSCOPY

Its basis is that a natural substance in the sample or fluorescent stain applied to the sample is excited by a light beam, emitting part of the absorbed energy as light beams.

Its most widely used application is to reveal fluorescence, such as in the detection of antigens and antibodies. Also, specific fluorescent molecules can be detected in the samples (GFP, etc.).

Fluorescence: as the excited molecules return to their normal state, they release the excess energy emitting visible light at a larger wavelength than the exciting radiation. Fluorescent objects appear illuminated against a dark background depending on the color of the stain used.

FLOW CYTOMETRY AND CELL SORTING

1. BD FACSCalibur Analysis Cytometer. 2 lasers with mechanical sorting module

Hardware

  • This device has two laser excitation sources, 488 nm (blue) and 635 nm (red), with simultaneous analysis capability of 4 fluorescence parameters, 2 scatter variables and time.
  • Doublets discrimination module for any fluorescence parameter derived from blue excitation.
  • Possibility of simultaneous threshold discrimination in two parameters.
  • Mechanical sorting module in a 50-ml tube.

Software

  • Data analysis system in Macintosh environment and Cellquest-Pro analysis software.

2. BD FACSCanto A Analysis System. Digital system with two laser sources, 488 nm (blue) and 633 nm (red) with automatic tube loader (BD FACSLoader)

Hardware

  • Allows the simultaneous detection of six fluorescence parameters, with the possibility of pulse area, height and width in different parameters simultaneously.
  • The laser beam signal is conducted to the collection system by optical fibers, being collected by an octagon (signals coming from blue excitation) or trigon (signal coming from red excitation) reflection system.
  • Possibility of using the (threshold) discriminator in two or more parameters simultaneously.
  • Automatic tube loader module.

Software

  • Windows environment with BD FACSDiva analysis software.

3. BD FACSAria III Sorting Cytometer. Digital system with three excitation sources: 488 nm (blue), 561 nm (yellow-green) and 633 nm (red). Single-cell sorting module in multi-well plates (cloning module). Integrated aerosol control module

Hardware

  • Allows the simultaneous detection of up to 12 fluorescence parameters, with the possibility of pulse area, height and width in several parameters simultaneously.
  • The laser beam signal is conducted to the collection system by optical fibers, being collected by octogons and trigons by a reflection system.
  • Allows the simultaneous sorting of four cell populations at the same time in different support types (tubes: 15 ml, 5 ml, eppendorf, different multi-well plates or slides).

Software

  • Data analysis in Windows environment with FACSDiva software and drop-delay automatic calculation system.
  • Other analysis software: Flow-Jo.

4. AutoMACS Magnetic Sorter (Miltenyi)

It performs a cell sorting process using a system of columns and antibodies conjugated to a magnet (immunomagnetic sorting). It allows sterile, automated flow since it is located inside a laminar flow cabin.

ADVANCED OPTICAL MICROSCOPY

1. LSM510 META Confocal Laser Scanning Microscope coupled to an Axiovert200 (Zeiss) inverted microscope

  • Axiovert 200 inverted microscope.
  • Motorized stage.
  • Incubation Module for in vivo assays (POC System).

Objectives

  • 10x 0.3 N.A. (Plan-Neofluar), no immersion.
  • 20x 0.5 N.A. (Plan-Neofluar), no immersion.
  • 40x 1.3 N.A. (Plan-Neofluar), immersion oil.
  • 40x 1.2 N.A. (C-Apochromat), water.
  • 63x 1.4 N.A. (Plan-Apochromat), immersion oil.

Optics

Clear field, phase contrast, Nomarski and fluorescence.

Laser lines

  • 405 nm diode (DAPI, Hoechst, Alexa405, Pacific Blue).
  • Argon 458/477/488/514 nm (CFP, GFP, YFP, FITC, Alexa488, Cy2).
  • Helium-Neon 543 nm (mCherry RFP, rhodamine, Texas Red, Alexa546, Alexa568, Cy3).
  • Helium-Neon 633 nm (IRP, Cy5, Alexa633, Alexa647).

Confocal Filters

  • 2 detection channels.
  • 1 Meta detector (polychromy detector, can also be used as third PM).

Software

  • Zeiss LSM510 vers.4.2. ZEN 2008 sp2, AxioVision vers.4.3, AxioVision 4, 3D Deconvolution module and Image-J.

2. Epi-fluorescence microscope

Olympus BX51 vertical epi-fluorescence microscope

Objetivos

  • 4x 0.13 N.A. (UPlanFl), no immersion.
  • 10x 0.3 N.A. (UPlanFl), no immersion.
  • 20x 0.5 N.A. (UPlanFl), no immersion.
  • 40x 0.75 N.A. (UPlanFl), no immersion.
  • 60x 0.65-1.25 N.A. (UPlanFl), immersion oil.
  • 100x 0.6-1.3 N.A. (UPlanFl), immersion oil.

Optics

Clear field, phase contrast, fluorescence and set of fluorescence filter cubes: BX-URA2 BX.

3. Epi-fluorescence microscope

Axio Observer A1 microscope.

Objectives

  • 5x 0.12 N.A. (A-Plan), no immersion.
  • 10x 0.25 N.A. (A-Plan), no immersion.
  • 20x 0.4 N.A. (LD Plan-NeoFl), no immersion.
  • 40x 0.6 N.A. (LD Plan-NeoFl), no immersion.

Optics

  • Clear field.
  • Phase contrast.
  • Fluorescence.
  • Set of fluorescence filter cubes:
    • EX G 365, BS FT 395, EM P 445/50 (DAPI, Hoechst).
    • EX BP 470/40, BS FT 495, EM P 525/50 (GFP, Alexa 488, FITC).
    • EX BP 530-585, BS FT 600, EM LP 615 (Cy3, IP).
    • EX BP 640/30, BS FT 660, EM BP 690/50 (Cy5).
    • Lighting system /iris X-Cite 120, fiber optics conductor.

Camera

  • AxioCam MRm Rev.3 monochrome high-resolution camera with FireWire.
  • RGB AxioCam ERc 5s digital microscope camera.

Software

  • AxioVision LE Fluorescence Lite module.

Manager

María Paz López Mato, immunologist(BIR)
mplopez@inbiomed.org - ext 240

She is the platform manager since 2010. She holds a Bachelor’s Degree in Fundamental Biology from the University of Navarre and a specialty (BIR) in Immunology. She completed her residency at the Marqués de Valdecilla University Hospital (1992-1994), during which time she was the manager of the flow cytometry area. Then she was hired by Becton Dickinson as Training Coordinator and application specialist for the Iberian Peninsula (1995-2001), providing technical support and user training activities on cytometry throughout Spain and Portugal. In 1997 she received the BD Bioscience European award to the best user trainer in flow cytometry.

Staff

Lorea Zabaleta
lzabaleta@inbiomed.org - ext 242
Idoia Gonzalez, PhD
igonzalez@inbiomed.org - ext 242

PUBLICATIONS

  • Etxaniz U, Pérez-San Vicente A, Gago-López N, García-Dominguez M, Iribar H, Aduriz A, Pérez-López V, Burgoa I, Irizar H, Muñoz-Culla M, Vallejo-Illarramendi A, Leis O, Matheu A, Martín AG, Otaegui D, López-Mato MP, Gutiérrez-Rivera A, MacLellan R, Izeta A. Neural-competent cells of adult human dermis belong to the Schwann lineage. Stem Cell Reports. 2014 Nov 11;3(5):774-88. doi: 10.1016/j.stemcr.2014.09.009. Epub 2014 Oct 16.
  • Iglesias JM, Leis O, Pérez Ruiz E, Gumuzio Barrie J, Garcia-Garcia F, Aduriz A, Beloqui I, Hernandez-Garcia S, Lopez-Mato MP, Dopazo J, Pandiella A, Menendez JA, Martin AG. The Activation of the Sox2 RR2 Pluripotency Transcriptional Reporter in Human Breast Cancer Cell Lines is Dynamic and Labels Cells with Higher Tumorigenic Potential. Front Oncol. 2014 Nov 4;4:308. doi: 10.3389/fonc.2014.00308. eCollection 2014.
  • Aguila JC, Blak A, van Arensbergen J, Sousa A, Vázquez N, Aduriz A, Gayosso M, Lopez Mato MP, Lopez de Maturana R, Hedlund E, Sonntag KC, Sanchez-Pernaute R. Selection Based on FOXA2 Expression Is Not Sufficient to Enrich for Dopamine Neurons From Human Pluripotent Stem Cells. Stem Cells Transl Med. 2014 Sep;3(9):1032-42. doi: 10.5966/sctm.2014-0011. Epub 2014 Jul 14.
  • Sáenz-Cuesta M, Irizar H, Castillo-Triviño T, Muñoz-Culla M, Osorio-Querejeta I, Prada A, Sepúlveda L, López-Mato MP, López de Munain A, Comabella M, Villar LM, Olascoaga J, Otaegui D. Circulating microparticles reflect treatment effects and clinical status in multiple sclerosis. Biomark Med. 2014;8(5):653-61. doi: 10.2217/bmm.14.9.
  • Rodriguez MS, Egaña I, Lopitz-Otsoa F, Aillet F, Lopez-Mato MP, Dorronsoro A, Lobato-Gil S, Sutherland JD, Barrio R, Trigueros C, Lang V. The RING ubiquitin E3 RNF114 interacts with A20 and modulates NF-κB activity and T-cell activation. Cell Death Dis. 2014 Aug 28;5:e1399. doi: 10.1038/cddis.2014.366.
  • Solaun MS, Mendoza L, De Luca M, Gutierrez V, López MP, Olaso E, Lee Sim BK, Vidal-Vanaclocha F. Endostatin inhibits murine colon carcinoma sinusoidal-type metastases by preferential targeting of hepatic sinusoidal endothelium. Hepatology. 2002 May;35(5):1104-16.
  • Alvarez-Domínguez C, Carrasco-Marín E, López-Mato P, Leyva-Cobián F. The contribution of both oxygen and nitrogen intermediates to the intracellular killing mechanisms of C1q-opsonized Listeria monocytogenes by the macrophage-like IC-21 cell line. Immunology. 2000 Sep;101(1):83-9.
  • Carrasco-Marín E, Paz-Miguel JE, López-Mato P, Alvarez-Domínguez C, Leyva-Cobián F. Oxidation of defined antigens allows protein unfolding and increases both proteolytic processing and exposes peptide epitopes which are recognized by specific T cells. Immunology. 1998 Nov;95(3):314-21.
  • Alvarez-Domínguez C, Vázquez-Boland JA, Carrasco-Marín E, López-Mato P, Leyva-Cobián F.Host cell heparan sulfate proteoglycans mediate attachment and entry of Listeria monocytogenes, and the listerial surface protein ActA is involved in heparan sulfate receptor recognition. Infect Immun. 1997 Jan;65(1):78-88.
  • Castaño J, Menendez P, Bruzos-Cidon C, Straccia M, Sousa A, Zabaleta L, Vazquez N, Zubiarrain A, Sonntag KC, Ugedo L, Carvajal-Verga ra X, Canals JM, Torrecilla M, Sanchez-Pernaute R, Giorgetti A. Fast and efficient neural conversion of human hematopoietic cells. Stem Cell Reports. 2014 Dec 9;3(6):1118-31. doi: 10.1016/j.stemcr.2014.10.008. Epub 2014 Nov 13.
  • Lang V, Aillet F, Da Silva-Ferrada E, Xolalpa W, Zabaleta L, Rivas C, Rodriguez MS. Analysis of PTEN ubiquitylation and SUMOylation using molecular traps. Methods. 2015 May;77-78:112-8. doi: 10.1016/j.ymeth.2014.09.001. Epub 2014 Sep 16.
  • Lang V, Pallara C, Zabala A, Lobato-Gil S, Lopitz-Otsoa F, Farrás R, Hjerpe R, Torres-Ramos M, Zabaleta L, Blattner C, Hay RT, Barrio R, Carracedo A, Fernandez-Recio J, Rodríguez MS, Aillet F. Tetramerization-defects of p53 result in aberrant ubiquitylation and transcriptional activity. Mol Oncol. 2014 Jul;8(5):1026-42. doi: 10.1016/j.molonc.2014.04.002. Epub 2014 Apr 13.
  • Gonzalez-Zubeldia I, Dotor J, Redrado M, Bleau AM, Manrique I, de Aberasturi AL, Villalba M, Calvo A. Co-migration of colon cancer cells and CAFs induced by TGFβ₁ enhances liver metastasis. Cell Tissue Res. 2015 Mar;359(3):829-39. doi: 10.1007/s00441-014-2075-6. Epub 2015 Jan 6.
  • Ibáñez E, Agliano A, Prior C, Nguewa P, Redrado M, González-Zubeldia I, Plano D, Palop JA, Sanmartín C, Calvo A. The quinoline imidoselenocarbamate EI201 blocks the AKT/mTOR pathway and targets cancer stem cells leading to a strong antitumor activity. Curr Med Chem. 2012;19(18):3031-43.

The Cytometry and Advanced Optical Microscopy Platform offers services to those external users who request them.

The priority objective of the platform is to offer a quality service and expertise to attain an optimal final result.

Each of the requested services includes consulting and discussion of technical aspects that the applicant must take into account to successfully carry out his/her experiment. Our aim is to offer a customized service to meet the user's needs; this is why following receipt of applications, an appointment will always be arranged with the potential user (in person, by mail or telephone) to work out all the details that will lead to an optimal execution of the experiment.

The platform also offers training courses for those users who want to start or carry out new applications in flow cytometry and advanced optical microscopy. Thus the Cytometry and Advanced Optical Microscopy Platform together with the Instituto de Investigación Sanitaria IDIVAL from Santander organizes annually a flow cytometry training course accredited by the Comisión de Formación Continuada de las Profesiones Sanitarias de Cantabria.

For details of the services offered by this platform, see the following e-brochure: PDF

GENERAL CONDITIONS

The Cytometry and Advanced Optical Microscopy Platform does not work with primary samples that entail biological risk or are infected with HIV or Hepatitis B or C.

Moreover, samples infected with amphotropic viruses (expression of fluorescent proteins) will not be processed until they have been kept for 5 days in post-infection culture.

Services offered by the Cytometry, Cell Sorting and Advanced Optical Microscopy Platform:

  • Multi-color phenotyping: up to 6 simultaneous fluorescences.
  • Viability and apoptosis assays.
  • Cell cycle and proliferation (BrdU, CFSE).
  • Intra-cellular cytokines.
  • Multiplex assays.
  • Metabolic assays.
  • Cell sorting of four simultaneous populations.
  • Cell sorting with cloning.
  • 3D reconstructions.
  • Multi-color confocal analysis.
  • 3D deconvolution.
  • Z-stacks.
  • Confocal analysis, in vivo studies.
  • Spectral overlap.

If you need any further information, please contact the platform manager María Paz López at mplopez@inbiomed.org

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