FLOW CYTOMETRY

Flow cytometry is a process in which physical and/or chemical characteristic of single cells or particles in suspension can be measured. In flow cytometry, measurements are made as the cells or particles pass through a flow cytometer, in a fluid stream. Flow cytometers are able to analyse multiple parameters in several thousand particles every second in "real time”. Flow cytometry can also be used to separate and isolate particles (sorting) with specified properties. A flow cytometer offers "high-throughput" (for a large number of cells) quantification of set parameters.

In a flow cytometer, a beam of light of a single wavelength (a laser light) is directed onto a hydro-dynamically-focused stream of fluid containing the cells or particles. Each particle or cell passes through one or more beams of light. The resulting light scattering combined with fluorescent emission (if the particle is labelled with one or more fluorochromes) provides multi-parametric analysis and information of the physical and/or chemical characteristics of single cells or particles flowing through.

APPLICATIONS:

• Simultaneous cell cycle and receptor analysis
• Surface/Intracellular antigens
• Small particle analysis (i.e. bacteria)
• Immunophenotyping
• Cell viability analysis
• Apoptosis
• Cell proliferation and activation
• Karyotyping
• Analysis of membrane potential and permeability

FLOW CYTOMETERS AVAILABLE

We currently have 4 flow cytometers located in lab 05 AW 00.

1. Becton Dickinson D FACScan flow cytometer analyser

This is used for flow analysis only. It has only one argon ion laser emitting at 488nm (blue laser), thus allowing two light scatter parameters (FSC & SSC) and three fluorescence channels to be measured. For less complex studies, this is a capable and reliable instrument.

Typical fluorescence that can be detected in each channel:

FL1:     FITC, YFP, GFP, Alexa 488, CFSE, Calcein, TO-PRO-3, TOTO-1, BODIPY

FL2:     PE, PI, Cy3, DiIC₁₈

FL3:     PerCP, Cy5, Cy7, PE-Cy5, PE-Cy7, 7-AAD, DRAQ5

Computer: Power Macintosh G4 running System 9.2

Software: CellQuest v3.3

2. Beckman Coulter Epics XL flow cytometer analyser

It is used for flow analysis only and it has only one argon ion laser emitting at 488nm (blue laser). Therefore, allowing two light scatter parameters (FSC & SSC) and three fluorescence channels to be measured. For less complex studies, this is a capable and reliable instrument.

Typical fluorescence that can be detected in each channel:

FL1:     FITC, YFP, GFP, Alexa 488, CFSE, Calcein, TO-PRO-3, TOTO-1, BODIPY

FL2:     PE, PI, Cy3, DiIC₁₈

FL3:     PerCP, Cy5, Cy7, PE-Cy5, PE-Cy7, 7-AAD, DRAQ5

Computer: running Windows 1998

Software: XL System II v 3.0 and EXPO32 ADC analysis software

3. Becton Dickinson FACS Canto flow cytometer analyser

This is used for analysis only and is equipped with 2 lasers, an argon ion laser emitting at 488nm (blue laser) and HeNe laser emitting at 633nm (red laser). The configuration is four colours from the blue laser and two colours from the red laser. Thus, allowing simultaneous analysis of six fluorescent parameters.

Typical fluorescence that can be detected in each channel:

FITC:FITC, YFP, GFP, Alexa 488, CFSE, Calcein, TO-PRO-3, BODIPY

PE:PE or PI, Cy3, DiIC18, PE-Texas Red

PerCP-Cy5.5:PerCP, PerCP-Cy5.5 or PI, PE-Cy5, 7-AAD, DRAQ5

PE-Cy7:PE-Cy7

APC:APC, Alexa 647

APC-Cy7:APC-Cy7

Computer: HP PC running Windows XP

Software: FACS DiVa v 5.0

4. Becton Dickinson FACS Aria flow cytometer cell sorter

Fluorescence-activated cell sorting (FACS) is a specialised type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument, as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest.

The FACS Aria is a high speed, high pressure, 4-way sorter. It has three solid state lasers emitting at 407nm (UV laser), 488nm (blue laser) and 633nm (red laser).  It is a digital system capable of high speed 4 ways and single cell sorting/analysis of up to 9 different fluorochromes.

Typical fluorescence to be detected in each channel:

FITC:FITC, YFP, GFP, Alexa 488, CFSE, Calcein, TO-PRO-3, TOTO-1, BODIPY

PE:PE or PI, Cy3, DiIC18, PE-Texas Red

PE-Texas Red:PE-Texas Red

PerCP-Cy5.5:PerCP, PerCP-Cy5.5 or PI, PE-Cy5, 7-AAD, DRAQ5

PE-Cy7:PE-Cy7

APC:APC, Alexa 647

APC-Cy7:APC-Cy7

Violet 1:Alexa Fluor 430

Violet 2:Pacific blue, DAPI, Hoechst, Alexa Fluor 405

Computer: HP PC running Windows XP

Software: BD FACS DiVa software v5.0

CELL SORTING APPLICATIONS:

• Transfected cells expressing GFP, YFP, CFP, TFP or BFP.
• Cell stably expressing GFP, YFP, CFP, TFP or BFP.
• Sub populations of cells with different phenotypes for downstream analysis.
• B and T cell subsets sorting.
• Cloning (single cell per well).
• Cell cycle fraction sorting (i.e. G0/G1, S, G2/M).

COST

All flow cytometers are available for use to all faculties on the University and external clients by prior arrangement. There is, however, a cost associated with running these instruments, please use the contact below for price information and rules for users.

BIOIMAGING

Confocal laser scanning microscopy (CLSM or LSCM) is a technique for obtaining high-resolution optical images with depth selectivity. The key feature of confocal microscopy is its ability to acquire in-focus images from selected depths, a process known as optical sectioning, and allowing three-dimensional reconstructions of topologically-complex objects. At the same time the out-of-focus light is suppressed, resulting in sharper images than those from conventional fluorescence microscopy techniques. CLSM permits to obtain images of planes at various depths within the sample allowing later 3D reconstruction. For this purpose, images are acquired by scanning point-by-point the three dimensional surface of an object by means of a focused laser beam and reconstructed with a computer. The biological specimens are labeled with fluorescent markers, allowing the multiple parameters detection.

Confocal microscopy provides the capacity for direct, non-invasive, serial optical sectioning of intact, thick, living specimens with a minimum of sample preparation as well as a marginal improvement in lateral resolution. Biological samples are often treated with fluorescent dyes to make selected objects visible. Also, transgenic techniques can create organisms that produce their own fluorescent chimera molecules (such as a fusion of GFP with the protein of interest).

INSTRUMENTS

We have one confocal microscope and a Zeiss inverted fluorescent microscope located in lab. 24 AY 02. The fluorescent microscope is in process of up-grading so it will be used for different applications that do not require confocal microscopy.

1. Zeiss LSM 510 META Confocal Microscope (on an Axiovert 200M Microscope)

• Fully automated inverted microscope.
• 3 independent lasers: argon (488nm), HeNe1 (543nm) and HeNe2 (633nm), with 6 laser lines including:  458, 477, 488, 514, 543 and 633 nm.
• META spectral detector - for spectral imaging – allows the easy differentiation of very similar fluorescence dyes emission and from auto-fluorescence. It allows up to 8 different fluorescence emission separations at the same time.
• Objective lenses: conventional 10x and 20x, and 40x oil DIC, 63x water and 63x oil DIC objectives.
• Enables study of live or fixed cells, as well as thicker specimens for co-localisation studies with multiple fluorescent probes as well as advanced techniques such as FRET, FRAP and spectral un-mixing.

Computer: running Windows 2000

Software: Zeiss LSM software v 3.2

2. Zeiss inverted fluorescent microscope (on an Axiovert 135 microscope)

• Manual fluorescent microscope.
• Objectives: conventional 10x (0.3NA) and 20x (0.5NA), and 40x (1.3NA) oil DIC, 63x (1.2NA) water, 63x (1.4NA) oil DIC objectives.
• Xenon arc lamp is used for fluorescence microscopy source instead of lasers, giving a wide choice of fluorophores, and is not limited to 2 or 3 specific excitation lines, as with lasers. Xenon illumination allows the excitation of UV and short wavelength blues dyes, such as DAPI and the Hoechst nuclear dyes.
• This microscope as well has 340 nm and 380 nm excitation filter cubes in the reflector turret and a wheel splitter for calcium ratio-metric studies

 COST

All instruments are available for use to all faculties on the University as well as external clients with prior arrangement. There is, however, a cost associated with running these instruments, please use the contact below for price information and rules for users.

CONTACT DETAILS

Dr. Alexandra Bermudez-Fajardo (in charge of the facility)
Biolmaging and Flow Cytometry Experimental Officer
Microbial Sciences Division
Faculty of Health and Medical Sciences
University of Surrey, Guildford, Surrey, GU2 7XH
Wolfson Cell Science Building (AW)
Room: 05 AW 01
Tel. office: 01483 689203
Tel. lab.: 01483 682506
Fax: 01483 686401
E-mail:a.bermudez-fajardo@surrey.ac.uk

Mrs Rebecca Holland
Technical support staff (for confocal)
Biochemical Science Division
Faculty of Health and Medical Sciences
University of Surrey, Guildford, Surrey, GU2 7XH
AY Academic Building
Room: 20 AY 02
Tel. office:01483 686387
Fax: 01483 686401
E-mail: r.holland@surrey.ac.uk

Dr. Ernesto Oviedo-Orta (academic lead for the facility)
Lecturer in Immunology
Microbial Science Division
Faculty of Health and Medical Sciences
University of Surrey, Guildford, Surrey, GU2 7XH
AX Academic Building
Room: 30 AX 01
Tel.: 01483 686424
Fax: 01483 686401
E-mail: e.oviedo-orta@surrey.ac.uk