Flow Cytometry Laboratory
Developed by: Instituto de Investigación Sanitaria Hospital La Paz
Flow cytometry has undergone significant advances within the research activity of IdiPAZ, and currently almost all groups involved in research at the Institute use these techniques. The equipment allows for phenotypic analysis of subpopulations, quantification of soluble factors in biological samples and culture supernatants, cell cycle experiments.
The Flow Cytometry Laboratory of IdiPAZ has the following facilities:
• One Beckman Coulter Navios cytometer analyser is a clinical system that delivers analytical excellence by coupling extraordinary sensitivity, resolution and dynamic range with high-speed data collection. With superior detection capabilities, the Navios provides accurate measurements, especially for rare events, which is critical to clinical diagnosis and monitoring. The fully-featured, easy-to-use Navios software provides tools to improve the quality of results as well as optimize workflow efficiencies in a clinical lab.
- This analyser has three lasers; Blue solid state Diode: 488nm, 22mwlaser output, Red solid state Diode: 638nm, 25mwlaser output and Violet solid state Diode: 405nm, 40mw laser output. The Navios can analyse ten colours simultaneously in samples marked with the following fluorochromes:• FL1: FITC, GFP, Cy2, Alexa 488 • FL2: PE, Cy3 • FL3: PI, ECD, PE-TEXAS RED, RED RFD • FL4: PE-Cy5, PerCP, Per-CP5.5, TC 7AAD, SPRD • FL5:PE-Cy7 • FL6: APC, Alexa 647 • FL7: APC-Alexa700, Alexa 700 • FL8: APC-Cy7, APC-Alexa750, APC-Alexa780 • FL9: Pacific Blue, eFluor 450 Cyan, Cyan, Violet RFD • FL10: Pacific Orange, AmCyan
- The Navios is connected to computer using the Navios acquisition and analysis programme.
• Two Becton Dickinson FACSCalibur™ cytometer analysers is a platform allows users to perform both cell analysis and cell sorting in a single benchtop system. The system supports a wide variety of research and clinical applications and is complemented by a broad suite of intuitive software solutions to streamline analysis for a wide range of applications including enumeration of lymphocyte subsets, stem cells, residual white blood cells, and reticulocytes.
- Each analyser has two lasers, a 488nm argon laser and a 635nm solid-state red diode laser. The FACSCalibur ™ can analyse four colours simultaneously in samples marked with the following fluorochromes: • FL1: FITC, Alexa 488 • FL2: PE, PI • FL3: PE-Cy5, PerCP, Per-CP5.5 • FL4: APC, APCCy7, Alexa 647
- The two cytometers are connected to Macintosh G4 OS.9.2.2 computers using the CellQuest™ acquisition and analysis programme, and Macintosh G5 OSX with CellQuestPro™ acquisition and analysis software
• The laboratory also has an additional Mac G5 OSX analysis station with CellQuestPro™ acquisition and analysis software
• The BD FCAP Array™ application (Windows XP/2000 version) is also available, which helps quantify soluble cytokines and proteins using flow cytometry.
The main applications of this technique are given in molecular and cellular biology and medicine are:
- Identification of cellular antigens by immunofluorescence techniques and the study of DNA content,
- Obtaining different phenotypic subpopulations,
- Quantification of soluble factors in biological samples and/or culture supernatants,
- Cell cycle experiments.