TYPES OF TECHNIQUES USED

The following techniques are carried out at our facilities.

1. Flow-cytometric quantification of immunophenotypic profiles: Preparation of biological samples for multicolour immunophenotyping of cell populations. This is performed using direct or indirect fluorescence techniques with monoclonal antibodies conjugated to fluorochromes for the characterisation of surface, intracellular or nuclear molecules that identify the phenotypic profile of populations of interest.
2. Fluorescence-based cell sorting (FACS ARIA II) Multicolour phenotyping allows us to isolate pathological populations that are present at very low levels in a sample. This is based on the optical characteristics of the equipment and on its fluidics, which allow the physical separation, by electrostatic methods, of a specific population of interest from the overall cell suspension.
3. Cell separation using the MACS immunomagnetic system: A column-based system in which the columns are placed inside a magnet. Cells are labelled with microbeads to identify epitopes of interest on the cell surface and are retained in the column. Populations that are not retained make up the negative fraction, containing cells without the ligand for the antibody used. This achieves high levels of purity. This automated system, based on coating the cells of interest with iron beads, enables their isolation while having minimal impact on the cells and maintaining high levels of viability.
4. Tissue dissociation: using mechanical, enzymatic or automated dissociation systems, including the GentleMACS tissue dissociator (Miltenyi Biotec), to obtain cell suspensions that can subsequently be characterised with monoclonal antibodies for flow-cytometric quantification.
5. Preparation of cell cultures for different procedures: functional studies by flow cytometry, metabolic studies (REDOX activity generation), and quantification of gene expression in living cells using fluorescent proteins.
6. Cultured cells for the study of extracellular microparticles and their involvement in pathological processes as a source of potential therapeutic targets.

TYPES OF ASSAYS

The flow cytometry and cell sorting laboratory performs, among others, the following assays: 

1. Multicolour analysis of surface and intracellular cell antigens.
2. Cell-viability assays: drug screening on cell lines. Identification of different stages of apoptotic cell death.
3. Cell-cycle analysis. Proliferation assays (CFSE).
4. Cell-signalling studies.
5. Functional studies (drug resistance, kinetic assays, cell-permeability studies, detection of intracellular generation of reactive oxygen species (ROS) in cell lines and tissue samples).
6. Reporter genes: analysis of fluorescent-protein expression efficiency.
7. Cell sorting using FACS or immunomagnetic isolation.
8. Detection of circulating tumour cells (CTCs) in peripheral blood and characterisation of antigen expression using multiparametric labelling.
9. Studies of extracellular macroparticles and/or exosomes by quantification in the small-particle module and fluorescence side scatter (SPD).

Samples analysed

At the Flow Cytometry Unit we process and analyse biological samples from clinical trials and patients, PBMCs isolated for research studies, cell cultures, tissue dissociation, etc., for the characterisation and/or isolation of cell populations of interest. We have established control samples for quantifying parameters in each procedure and/or working protocol for each requesting user.

Requests must be submitted by completing the corresponding request form, which will be available on the Unit website:

• Data Analysis Service request form
• Flow Cytometry Service request form
• Cell Sorting Service request form

Sample-receipt conditions will be taken into account, including correct sample identification and presentation conditions, with particular consideration given to sample viability.

The information to be provided by the researcher in order to carry out an analytical flow-cytometry study is:

• Experimental design and number of samples.
• Protocol to be carried out.
• Cell type to be analysed.
• Combination of fluorochromes to be used in the experiment.

When requesting cell sorting of samples of interest, researchers must provide the following information:

• Experimental design.
• Protocol to be carried out.
• Cell type to be sorted.
• Number of samples to be sorted.
• Identification of the cell populations to be sorted: by fluorescence emission from the fluorochromes used (in the case of the FACSAria II sorter) or by immunomagnetic beads (when using the AUTOMACS PRO separator).
• Cell concentration of the sample to be sorted.
• Method for collecting the sorted cells (type of tube).
• Whether sterile conditions are required during sample processing.

The necessary cleanliness levels are maintained on all equipment and in the laboratory for assays requiring sterile conditions.

Data analyses are performed comparatively between control samples and samples of interest using standardised analysis software that is widely accepted internationally.

FLOW CYTOMETRY AND CELL SORTING UNIT FEES

Current fees