- Renan Antonialli
- Julia García
What is Flow Cytometry?
Flow Cytometry is a specialized way of analyzing cells or particles, in suspension, as they pass in a single file through a sensing point (1 or more laser beams) where several measurements related to the physical and chemical characteristics of each cell are taken and quantified via an electronics system. It allows for multiple measurements on large numbers of individual cells in a short time, making flow cytometry a very useful a powerful tool when looking for rare and specifically-defined populations, so its importance in the cancer research field. We could also isolate those specifically-defined populations through cell sorting.
The Flow Cytometry Core Unit is equipped with state of the art instrumentation, both analysers and high-speed cell sorters, that allow us to analyze and retrieve cells or particles of interest based on their pattern of expression. Our aim is to offer an extensive, flexible and user friendly service and to actively try to develop and establish new methods and techniques, involving the use of flow cytometry, of interest to CNIO researchers.
Currently our unit is equipped with 4 analytical cytometers and three cell sorters:
- 1 FACS Calibur (Becton Dickinson): dual-laser analyzer up to 6 parameters detection, equipped with 488nm and 635nm lines. It runs with CellQuest v5.1.1.
- 2 FACS Canto II (Becton Dickinson): a 3 solid-state laser analyzer capable of up to 10 parameters detection, equipped with 405nm, 488nm and 633nm lines. The current configuration allows for the detection of 4 fluorescent parameters out of the 488nm line and 2 out of the 633nm and 405nm respectively. It is also equipped with a High Throughput System (HTS module) that runs 96- and 384 microtiter plates. It runs with FACS Diva v6.1.3.
- 1 LSRII FORTESSA (Becton Dickinson): 4 laser analyzer up to 17 parameters detection in its current configuration, equipped with 355nm, 488nm, 561nm and 640nm lines. The current configuration allows for the detection of 5 fluorescent parameters out of the 488nm and 561nm lines, 3 out of the red line and 2 out of the UV line. It runs FACS DIVA software v6.1.3.
- 2 COUNTESS (Invitrogen): slide-based cell counter that uses Trypan Blue exclusion to quantify viability. It takes up to 10uls of culture.
- 1 AutoMACS Pro (Miltenyi): Automated column based magnetic cell separator, allow for multisample labelling and separation of various cell types using magnetic particle conjugated antibodies that will bound to the cell of interest (positive selection) or to the unwanted cell population (depletion) allowing for enrichment of cell subsets in a short time.
- 1 FACS ARIA IIu (Becton Dickinson): high-speed 4-way digital cell sorter capable of detecting up to 12 parameters. Equipped with a 407nm, 488nm and 633nm excitation lines and with a single cell deposition unit allowing for single cell cloning. We can sort into various receptacles including 5ml, 15ml Falcon tubes, 6-, 24-, 48-, 96- and 384-well plates, slides and another custom containers. It runs with FACS Diva v6.
- 1 InFlux (Cytopeia-Becton Dickinson): high-speed 4-way in-air stream cell sorter capable of detecting up to 18 parameters in its current configuration. Equipped with a 355nm, 488nm, 561nm and 640nm excitation lines and with a single cell deposition unit allowing for single cell cloning. We can sort into various receptacles including 5ml, 15ml Falcon tubes, 6-, 24-, 48-, 96- and 384-well plates, slides and another custom containers. It runs with Sortware v10.0.0.650. It is installed into a biological safety cabinet so we can accept Hazard Class II samples for cell sorting.
- 1 MoFlo ASTRIOS (Beckman Coulter): high-speed 6-way in-air stream cell sorter capable of detecting up to 18 parameters in its current configuration. Equipped with a 355nm, 488nm, 561nm and 640nm excitation lines and with a single cell deposition unit allowing for single cell cloning. We can sort into various receptacles including eppendorfs, 5ml, 15ml Falcon tubes, 6-, 24-, 48-, 96- and 384-well plates, slides and another custom containers. It runs with Summit v6.
- 1 OrtoGentleMACS (Miltenyi) bench-top instrument for standarized automated gentle tissue dissociation and sample preparation.
The unit also counts with specialized software packages to analyze and quantify the cell subsets, including licenses of FlowJo, ModFit and FCS Express.
The following techniques involving flow cytometry are currently in use:
- Multicolor analysis (surface immunophenotyping and intracellular staining)
- Apoptosis analysis (Annexin V, TUNEL, TMRE, enzyme activity, SubG1 detection of cellular DNA)
- Cell cycle analysis (BrdU and EdU incorporation, DNA content analysis)
- Functional analysis (Calcium flux, CFSE, ROS production)
- Phosphoproteins detection (Phospho Histone-3, p38, Jnk, Stat5) in cell lines and primary cells from tissue suspensions
- Cell sorting of established cell lines and primary samples both mouse and human.
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