Cytometry



1.    FLOW CYTOMETRY FACILITY

2.    EQUIPMENT
3.    APPLICATIONS
4.    FACILITY
5.    RATES 
6.    BIBLIOGRAPHY
7.    EDUCATIONAL WEB PAGES



CYTOMETRY



1.    FLOW CYTOMETRY FACILITY

Flow cytometry is a powerful technique for the analysis of multiple parameters of individual cells within heterogeneous populations. The flow cytometers performs this analysis by passing thousands of cells per second through a laser beam and capturing the light that emerges from each cell as it passes through. The data gathered can be analyzed statistically by flow cytometry softwares to report cellular characteristics such as:
•    Cell size (FSC)
•    Cell complexity (SSC)
•    Fluorescence

2.   EQUIPMENT

The Flow Cytometry Core Facility of the Andalusian Center for Developmental Biology is equipped with the following instruments:

      
 
1.   BD FACSAria Cell Sorter.

       •    two lasers: 488 nm y 633 nm
       •    9 parameters: FSC, SSC and 7 fluorescence colors
       •    Sample acquisition with temperature control
       •    Acquisition rates of up to 70 000 events per seconds
       •    Sorts of up to four populations simultaneously
       •    Cell sorting in tube or culture plate
       •    Nozzles: 70 and 100 micron
       •    Software: FACSDiva


The available optic configuration is detailed below:
 
Laser PMT LP Mirror BP Filter Fluorocromos
Blue 488 nm A 735 780/60 PE-Cy7
B 655 695/40 PerCP-Cy5.5 ó PI
675/20 PerCP ó PE-Cy5
C 595 610/20 PE-Texas Red
D 556 576/26 PE ó PI
585/42 PE/PI
E 502 530/30 FITC
F   488/10 SSC
Red 633 nm A 735 780/60 APC-Cy7
B 685 712/21 APC-Cy5.5
C 660/20 660/20 APC

      
2.    BD FACSCalibur

       •    Laser 488
       •    5 parameters: FSC, SSC and 3 fluorescence colors
       •    Cell Sorting
       •    Software: Cell Quest Pro



 



The available optic configuration is detailed below:
 
Laser PMT LP Mirror Filter Fluorochromes
BLUE
488 nm
FL1   530/30 FITC
FL2 560 585/42 PE
FL3 640 650 LP PerCP
    488/10 FSC, SSC


      
 
3.   SONY MA900 Cell Sorter.

       •    Four lasers on two axes: 488/561nm and 405/638 nm
       •    14 parameters: FSC, BSC and 12 fluorescence colours
       •    Sample acquisition tube types: 0,5ml, 1.5ml, 5ml and 15ml
       •    Acquisition rates of up to 70 000 events per second
       •   Temperature control of sample and collection tubes: 5oC and 37oC
       •    Cell sorting in 2-way and 4-way tubes, epp or 6,12,24,48,96 & 384- well plates
       •    Index sorting software
       •    Nozzles: 70 and 100 micron
       •    Software: SONY-MA (online/offline)
 


The available optic configuration is detailed below:

 
Laser  Optical filters LPF BP Filter Fluorochromes
FL6 450/50 Dapi, Bviolet 421
FL7/LP10 487.5LP 525/50 BViolet510,Pacific Orange
FL8/LP4 561LP 585/30 BViolet570
405nm FL9/LP3 600LP 617/30 BViolet 605
FL10/LP7 639LP 665/30 BViolet 650
FL11/LP6 685LP 720/60 BViolet 711
FL12/LP5 752LP 785/60 BViolet 785
FL1 525/50 Fitc, AlexaFluor 488,EGFP,EYFP
FL2/LP4 561LP 585/30 PE
488nm FL3/LP3 600LP 617/30 PE-Texas Red, PI,
FL4/LP7 639LP 695/50 7AAD, PE-cy5, PerCP
FL5/LP1 752LP 785/60 PE-CY7
BSC 488/17B
FL2/LP4 561LP 585/30 mOrange, DsREd-Monomer, tdTomato
561nm FL3/LP3 600LP 617/30 mCherry, Alexa Fluor 594
FL4/LP7 639LP 695/50 mPlum
FL10/LP7 639LP 665/30 APC, Cy5, Alexa Fluor 647
638nm FL11/LP6 685LP 720/60 APC-Cy5.5, Alexa Fluor 700
FL12/LP5 752LP 785/60 APC-Cy7, APC-Alexa Fluor 750


3.    APPLICATIONS

Flow cytometers have the ability to measure multiple celular parameters and markers of a large number of cells at a rapid rate. So the applications of flow cytometry are numerous, and this has led to the widespread use of these instruments in biological research.
The main applications that we have developed in the center are:
•    Cell viability assays
•    Membrane potencial
•    Inmunophenotyping
•    Cell cycle analysis
•    Apoptosis and necrosis
•    Cell sorting
      o    Stem cells (mouse)
      o    Cell lines
      o    Drosophila discs cells (eyes and wings)
      o    Hematopoietic cells (mouse blood, kidney, liver, bone marrow, spleen, thymus)

4.   FACILITY 

4.1    Contact with Corín Díaz Ramos
         Telephone: 95 4977432
         E-mail: cdiaram@upo.es
4.2    Sample preparation: Samples must be prepared on aseptic conditions and must be filtered with a  40µm strainer. We recommend discussing previously the experimental design with the facility's staff.
4.3    Service:
         - Assisting researchers in designing experiments
         - Analytical cytometers' user training
         - Assisting researchers with analysis and data interpretation
         - Sorting cell populations


 
5.    RATES
 
  Services offered UPO/CSIC Universities/OPIs Private
  FACSCalibur analysis 10€ 15€ 20€
  FACSAria analysis 15€ 22,5€ 30€
  FACSAria cell sorter 20€ 30€ 40€
  Sony analysis 15€ 22,5€ 30€
  Sony cell sorter 20€ 30€ 40€


6.    BIBLIOGRAPHY


1.    Flow cytometry and cell sorting (1992) A. Radbruch 
       Springer-Verlag, Berlin.
       (579 FLO)

2.    In living color: protocols in flow cytometry Rochelle A. Diamond, Susan DeMaggio
         (579 IN)

3.    Flow Cytometry. A Practical Approach (2000) Ormerod, MG.
       3rd edition. Oxford University Press, Oxford. Robinson, J.P.,
       (576 FLO)

4.    Practical Flow Cytometry (2003) Howard Shapiro.
       Fourth Edition  Wiley-Liss, New York
       (576 SHA)

5.    Cytometry: new developments (2004)
       San Diego: Elsevier
       (576 CYT)

6.    Flow cytometry protocols (2004)
       Totowa, New Jersey, Humana Press, cop.
       (576 HAW flo)

7.    Flow cytometry: principles and applications (2007)
       Totowa, New Jersey, Humana Press, cop.
       (576 FLO)



7.    EDUCATIONAL WEB PAGES


1. Invitrogen: Fluorescence Tutorials
www.invitrogen.com/site/us/en/home/support/Tutorials.html

2. Invitrogen: Spectrum Viewer
http://probes.invitrogen.com/servlets/spectraviewer

3. BD Biosciences: Spectrum Viewer
www.bdbiosciences.com/colors/fluorescence_spectrum_viewer

4. Flow Cytometry Core Facility. EMBL
http://www-db.embl.de/jss/EmblGroupsHD/g_251

5. Pardue University Cytometry Laboratories
www.cyto.purdue.edu/

6. Iberian Cytometry Society
www.citometria.org/

7. Compensation tutorial
http://drmr.com/