Servicio de Citometría

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
Azul 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
Rojo 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.    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 Katherina García
         Telephone: 95 4977432
         E-mail: kgargar@upo.es
4.2    Sample preparation: Samples must be prepared on aseptic conditions and must be filtered with a          70µm strainer.
4.3    Service:
         - Assisting researchers in designing experiments
         - Training of users in the use of analytical cytometers
         - Assisting researchers with analysis and interpretation of data
                  - Sorting cell populations

5.   RATES 

 

Cytometer	CSIC/UPO	OPI	Private
FACSAria	20€	30€	40€
FACSCalibur	10€	15€	20€

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/