Flow Cytometry Unit

Flow cytometry is a method, somewhat analogous to fluorescent microscopy, in which measurements are performed on cells in liquid suspension, which flow one at a time through a focused laser beam at rates up to several thousand particles per second. Light scattered and fluorescence emitted by the cells is collected, filtered, digitized and sent to a computer for analysis. Practically all of the work done at the CABIMER flow cytometry facility involves measuring the binding of a fluorochrome-labeled probe to cells and the comparison of the resultant fluorescence to the background fluorescence of unstained cells. Another service at CABIMER of the flow cytometry facility is cell sorting, in which the cells are separated and recovered from suspension based upon properties measured in flow. Cells that are recovered via flow sorting are viable and can be collected under sterile conditions. The facility can most often provides subpopulations that are in excess of 99% pure.

Staff

Scientific Manager
Abelardo López Rivas ( This e-mail address is being protected from spambots. You need JavaScript enabled to view it )

Technical Managers

María José Quintero ( This e-mail address is being protected from spambots. You need JavaScript enabled to view it )

Equipment

The Flow Cytometry Facility is equipped with two BD FACSCalibur analyzers and a BD FACSAria sorter.

Cell analysis
The BD FACSCalibur system is a four-color, dual-laser, benchtop system designed to support a wide range of both basic and clinical research. The instrument can detect three fluorochromes excited by the 488nm argon-ion laser and one fluorochrome excited by the 635nm red-diode laser. Examples of fluorochromes that can be analyzed on this instrument include:
  • 488nm excitable: FITC, PE, PerCP, PE-Cy5, PE-Cy5.5, PE-Cy7, PerCP-Cy5.5, CFSE, GFP, Alexa 488, PI, 7-AAD, DRAQ 5, Acridine Orange, Fura Red, Fluo-3, Cy3, JC-1.
  • 635nm excitable: APC, DRAQ 5, Cy5, Alexa 633, Alexa 647.

The emission filters are static, and are as follows:
  • Emissions from the 488nm excitation
    • FL1-530/30 nm (515-545 nm)
    • FL2-585/42 nm (564-606 nm)
    • FL3-670 nm LP (>670 nm)
  • Emissions from the 635 nm excitation
    • FL4-661/16 nm (653-669 nm)

 Cell sorting

The facility is also equipped with a BD FACSAria sorter. The BD FACSAria is a benchtop sorter that has three air-cooled lasers at 407, 488 and 633 nm allowing multicolour analysis of up to 15 parameters. The FACSAria can sort 1, 2, 3 or 4 populations simultaneously and is equipped with an Automated Cell Deposition Unit for sorting into multiwell plates or slides. The FACSAria is equipped with a refrigeration system that allows cooling of the sort sample as well as the sort collection tubes. It can accept various sample input tubes including 1 ml microtubes, 12x75-mm test tubes and 15 ml tubes into the sample and collection chambers.


Frequent applications

  • Detection of cell surface antigens by immunofluorescence (immunophenotyping).
  • Identification of reporter genes through fluorescence’s dyes.
  • Cell viability assays.
  • Cell toxicity by changes of membrane potential
  • Cell cycle analysis (Propidium Iodide).
  • Apoptosis and necrosis.
  • Characterization of signalling in transfected cells with GFP, RFP (green, red fluorescent proteins).
  • Oxigen reactive species analysis (superóxide, peroxide and Nitric oxide).
  • Changes in intracelular pH and Ca2+ flux with specific fluorochromes.
  • Cellular proliferation measured by the dilution of the fluorescent dye CFSE with each round of division.


Service rates

For information about rates, contact with the staff of the Unit. ( This e-mail address is being protected from spambots. You need JavaScript enabled to view it )