Cytometry Unit

The imaging cytometer acquires up to 12 images simultaneously from each cell/object. The signal is collected in a CCD camera that operates in TDI (Time Delay Integration) mode generating a combined image from all the previous ones. The ImageStream imaging cytometer combines 4 lasers (collinear 2 to 2), with 2 CCD cameras that collect the fluorescence spectrum, plus a microscope with 20X, 40X and 60X objectives. The acquisition volume is between 20-200 µl with an efficiency of 95% sample utilization. The optical configuration can be seen in Table 1 and 2.

Samples should be purchased in 1.5 ml tubes and at a concentration of at least one million cells in 50 µl.

• What cells can be analyzed?

Both fresh and fixed cells can be acquired. Cell size should be less than 120µm for 20x magnification, 60µm for 40x and 40µm for 60x. This cytometer can detect objects as small as 0.5 µm macroparticles, bacteria and platelets using the optical surface and SSCA parameters (complexity). Small objects with sizes of 100 nm – 50um can be detected based on fluorescent markers.

• How long does cell analysis take?

Depending on the concentration of cells and the frequency of the population of interest, it can take from a few minutes to a few hours to record the data for the number of cells to be acquired. The acquisition speed varies depending on the objective used, it can be between 1,200-4,000 cells/sec. The subsequent analysis of the sample is performed in IDEAS software.

• Is it necessary to book in advance?

The equipment is only used by the specialized staff of the Unit. There is the possibility of assisted and independent reservation (internal users only). Independent use is available during the working hours of the unit’s technicians, depending on the availability of the reservation. And it can be done with prior training by the Unit to the user.

Channels: 135, mass range: 75-209 amu.

Sensitivity: < 0.3% for 160Gd (M+1 for 159Tb), < 0.3% for 174Hf (M+1 for 175Lu).

Instrument response: 400K to 600 K counts/pg 159Tb.

Limit of detection: 350 antibodies/cell.

Peak throughput: 500 (events/sec)

Flow rate: 30µl/min

The system is fast and simple, very similar to that performed with fluorochromes. Cells in suspension are also labeled in tubes with a panel of antibodies to the protein of interest. More than 1000 antibody conjugates are available for human and mouse samples covering a wide range of applications including phenotyping, immune response monitoring, cytokine expression, signaling responses, apoptosis, structure and cytoskeleton, enzyme activity and cell cycle, immuno-oncology, as well as more than 20 pre-validated kits for different human and mouse applications. In addition, antibody labeling kits allow labeling of most monoclonal antibodies (IgG) of your choice with more than 50 different metals. Tools for panel design are available.

There is also the possibility to use a Barcoding system that allows to analyze a pool of up to 20 samples. Thus samples with different barcodes can be combined, subsequently marked and acquired as a multiplexed sample. Subsequently the software will debarcode and separate each individual sample.

It is very important to use metal-free buffers. The last step of the protocol includes a nucleic acid intercalant used to identify events. There are several general protocols already validated for the labeling of the three cellular compartments (surface, cytoplasm and nucleus). Samples can be marked and sent for further analysis.

https://pdv2.dvssciences.com/login

https://www.standardbio.com/support/instrument-support/cytof-helios-support#documents-anchor

https://iti.stanford.edu/himc/immune-monitoring-virtual-course-2020/part-2.html

• What cells can be analyzed?

Both fresh and fixed cells, both human and mouse, can be acquired. It is also possible to work with nanoparticles or any element that can be identified by mass spectrometry. The application areas are shown in the following table.

• How long does cell analysis take?

Depending on the concentration of cells and the frequency of the population of interest, it can take from a few minutes to a few hours to record the data of the number of cells we want to acquire.

The system generates files in .fcs format that can be analyzed in conventional and/or specific cytometry software for this type of complex systems.

http://fluidigm.cytobank.org/

https://astrolabediagnostics.com/

https://www.flowjo.com/

http://www.omiq.ai/

https://iti.stanford.edu/himc/immune-monitoring-virtual-course-2020/part-3.html

• Is it necessary to book in advance?

No. The equipment is only used by the Unit’s specialized personnel.

Two cytometers are available for use in conventional cytometry.

Samples should be placed in 5 ml tubes compatible with BD.

• What cells can be analyzed?

Both fresh and fixed cells can be acquired. This cytometer can detect objects as small as 0.5 µm microparticles, bacteria and platelets using optical surface parameters and SSCA (complexity). Small objects with sizes up to 250 µm can be detected based on fluorescent markers.

Applications are focused in the areas of cell differentiation, immunology and pathology where, in addition to working with primary cells and cell lines, detection of the expression of reporter genes marked with fluorescent proteins can be added to monitor the efficiency of transfections. Likewise, assays can be performed to provide information about molecular interactions, protein structure and DNA sequence. Study of nanoparticles and microvesicles.

• How long does cell analysis take?

Depending on the concentration of cells and the frequency of the population of interest, it can take from a few minutes to a few hours to record data on the number of cells we want to acquire. The acquisition speed varies depending on the target used, it can be between 10-100 µl/min with an acquisition rate of 10,000 events/sec. Subsequent sample analysis is performed in FACSDiva software or cytometry analysis software. The software available at the Unit are FlowJo and OMIQ.

• Do I need to book in advance?

The equipment is only used by the specialized staff of the Unit. There is the possibility of assisted and independent reservation (internal users only). Independent use is available during the working hours of the unit’s technicians, depending on the availability of the reserve. And it can be done with prior training by the Unit to the user.

The sorter is integrated in a class II type A2 biological safety cabinet to protect the samples and the user from contamination. It has the possibility of sorting with 70-, 85-, 100-, and 130-µm Nozzel. Hot and cold collection system for 2-4 samples at the same time. Single cell sorter system in 6, 24, 48, 96 and 384-well plates.

Capable of sorting bulk cells into 1.5 ml Eppendorf, 5 ml FACS tubes or 15 ml Falcon tubes, as well as sorting single cells onto plates or slides. For sample preparation for in-house users, please refer to the recommendations in the document

https://afrodita.genyo.es/resources/Centro/Citometr%c3%ada/preparacionMuestrasSorter.pdf

• What cells can be analyzed?

Both fresh and fixed cells can be acquired. This cytometer can detect objects as small as 0.5 µm microparticles, bacteria and platelets using optical surface parameters and SSCA (complexity). Small objects with sizes up to 250 µm can be detected based on fluorescent markers.

Separations of homogeneous cells or cell populations are performed in order to subsequently carry out biochemical, molecular or cell differentiation assays of populations of interest such as the following:

– Their subsequent expansion and enrichment in culture.

– Use them for functional assays.

– Use the separated cells for transplantation in animals or patients.

– Separating X from Y spermatozoa for sex selection.

– Separating yeast, bacteria or phytoplankton.

– Selection and separation of subcellular organelles.

– Isolation of specific chromosomes, currently the only technique that allows the practical isolation of a large number of chromosomes.

– Isolation of a sufficient number of cell subpopulations for use in genomic analysis (microarrays).

• How long does cell analysis take?

The speed of cell sorting depends on several factors: The initial concentration of the sample, the frequency of cells of interest to be sorted from the initial sample, the type of cell being sorted, the quality of the cells provided in the sample and the differential pressure applied to the cells. This means that cell sorting speeds are variable and are determined on a case-by-case basis.

However, as an example: to pass 1mL of PBMCs at a concentration of 10 million cells/mL at the maximum differential pressure (highest speed) will take approximately 30 minutes.

Users are strongly advised to filter their cells prior to sorting and to suspend their samples in EDTA-containing solution to avoid cell clumping. Cell clumping can clog cell sorters and prolong sorting time. For more sample preparation tips go to the following document:

https://afrodita.genyo.es/resources/Centro/Citometr%c3%ada/preparacionMuestrasSorter.pdf

• Do I need to book in advance?

No. The equipment is only used by the specialized personnel of the Unit.

The lúminex 100 System allows the analysis of up to 100 immunoassays, gene complexes and enzyme assays per well. It supports various assay formats, including nucleic acid assays, receptor-ligand assays, immunoassays and enzyme assays.

The emission spectrum of each of these beads is unique, enabling simultaneous identification of all beads and therefore the reaction or assay being carried out on the surface of each bead and all of these procedures within the same reaction tube. The microspheres are labeled with two fluorophores in such a way that a ratio of both is established in each one of them. Thus we will have the first of the microspheres with 100% of the first fluorophore and 0% of the second, the 2nd microsphere with 99% of the first fluorophore and 1% of the second, the 3rd with 98% of the first fluorophore and 2% of the second and so on until completing 100 perfectly identifiable microspheres within the same reaction tube. Subsequently, the microspheres are forced through a flow stream.

Each bead is sorted according to the ratio of its internal fluorescent labeling. In addition to this, the lúminex 100 System scans each of the microspheres to determine the presence or absence of another fluorophore, which may or may not have bound to the specific assay reagents (antibodies, oligonucleotides, substrates, etc.) anchored on the surface of the spheres. These spheres are polystyrene particles that have undergone crosslinker action during polymerization to achieve high thermal and physical stability. To ensure the stability of these microspheres it is essential to protect them from light and high temperatures.

Acquisition is performed in a 96-well plate producing up to 9,600 data points in about 35 minutes. It is capable of providing fast and cost-effective bioassay results.

The Bio-Plex 200 system can test a variety of sample types, including serum, plasma, culture medium, extracts, milk, saliva, and others.

It is important to prepare samples consistently and use established protocols for proper immunoassay performance, regardless of the sample source. Samples should be tested soon after collection or frozen in aliquots to avoid multiple freeze-thaw cycles, which can degrade the sample.

Serum samples should be free of lipemia, hemolysis, platelets and precipitates. Some samples, such as tissue homogenates, may require ultracentrifugation for clarification due to high levels of debris.

• What cells can be tested?

You can test various types of samples, such as serum, plasma, culture medium, extracts, milk, saliva and others. A wide variety of kits are currently available. Kits for measurement of: IgG1, IgG2, IgG3, IgG4, IgA, IgM and IgE, Hepatitis B, SNP’s, HIV, CV and HSV viral load, DNA typing, Transcription profiling, Cytokine measurement, Epitope mapping, Cardiac markers, Drugs of abuse, Allergy and autoimmunity testing, hCG, AFP and ToRCH, etc.

• How long does cell analysis take?

One 96-well plate and a throughput of up to 9,600 data in approximately 35 minutes.

• Do I need to book in advance?

The equipment is only used by the specialized staff of the Unit, except for internal users with prior training by the Unit.

Manufacturer: BD Biosciences
Technical characteristics: It has a Coherent® Sapphire™ air-cooled solid-state blue laser (488 nm and 20 mW power), which can detect four colours. A JDS Uniphase™ HeNe red laser (633 nm and 17 mW), for two colours. The total number of detectors is 8 (6 fluorescence detectors and two morphological parameter detectors). The software for sample acquisition and analysis is FACSDiva. The system has a quality module to optimise voltages and to control and monitor the operation of the system.

Manufacturer: BD Biosciences
Technical characteristics: BD FACSVerse™ is a cytometer for research use, it has three laser lines, 488 nm (blue) for 4 colours; 633 nm (red) for two colours and violet (407 nm) for two colours. It can detect up to 10 scattering parameters (8 fluorescence detectors and two morphological parameters). It features a laser system that minimises light loss and maximises resolution for multi-parametric applications. The lasers feature a micromotor for automatic alignment. BD FACSVerse™, in addition to the standard acquisition speeds (high, medium and low), features a high sensitivity mode that makes it easier to detect weakly labelled particles. Its fluidic system allows manual sample acquisition in any tube format. It has a sample loader that allows to analyse from 30 to 40 tubes in a single run, up to 96 and 384-well plates. It has a volumetric meter. The software for sample acquisition and analysis is FACSSuite. The system has a quality module that optimises voltages and controls and monitors the operation of the equipment, which provides high reproducibility of the experiments.

Manufacturer: BD Bioscencies
Technical characteristics: It has three laser lines: The Coherent® Sapphire™ blue solid-state diode (488 nm and 13 mW of power), for detecting five colours; red laser (633 nm and 11 mW JDS Uniphase™ HeNe air-cooled), for two colours and the violet solid-state laser (407 nm and 10 mW) for two colours. In total, it can detect up to 11 scatter parameters at the same time (9 fluorescence detectors, Forward scatter (FSC) and Side scatter (SSC)), as well as purify different types of cell populations based on certain characteristics. The acquisition speed can reach up to 70000 events per second. It supports different supports for both sample acquisition and sample separation. It is equipped with an ACDU (Automated Cell Deposition Unit) for separation in multiwell plates or on slides. It also has a cooling system for temperature-controlled sample acquisition and separation. It uses FACSDiva software for sample acquisition and analysis.

Manufacturer: BD Bioscencies
Technical characteristics: It has four laser lines: The Coherent® Sapphire™ blue solid-state diode (488 nm and >50 mW power), which detects two colours; red laser (640 nm and 100 mW JDS Uniphase™ HeNe air-cooled) for two colours, green laser (561 nm and >50 mW) for five colours and the violet solid-state laser (405 nm and 85 mW) for six colours. In total, it can detect up to 18 scattering parameters at the same time (16 fluorescence detectors, Forward scatter (FSC) and Side scatter (SSC)), as well as purify different types of cell populations based on specific characteristics. The acquisition speed can reach up to 70000 events per second. It supports different supports for both sample acquisition and sample separation. It is equipped with an ACDU (Automated Cell Deposition Unit) for separation in multiwell plates or on slides. The equipment is equipped with a Type II Biological Safety Cabinet and an AMU aerosol aspiration system. The sample can be refrigerated during acquisition and separation. It uses FACSDiva 8.0 software for sample acquisition and analysis.