Faculty of Science - Leading creativity and innovation in the sciences


Flow Cytometry Centre


The Flow Cytometry Centre provides researchers with technical expertise, training and access to instrumentation to enable and support the use of a wide range of flow cytometry techniques.  Flow cytometers are able to analyse several thousand cells (particles) every second, in "real time," and specialised cell sorting cytometers can actively separate and isolate cells (particles) having specified properties. Flow cytometry, typically using fluorescent probes which bind to specific cell associated molecules, allows measurements of various molecular characteristics of individual cells (or particles) suspended in a fluid stream.

The use of flow cytometry can be divided into two broad categories, analysis and cell sorting.

Analysis

Analyses can be performed on either our 4 colour BD FACS Calibur, our 4 colour BD Accuri C6 or our 17 colour BD SORP FACS Aria II cell sorter. The FACS Calibur is largely used for basic immunophenotyping, or single colour detection (see applications below). The BD Accuri C6 is also a 4 colour analyser which has an automated plate sampler and is therefore the machine of choice for running cytometric bead arrays or any other high throughtput assays. The FACS Aria II is not only used for cell sorting, but also highly complex multi-parametric cell analyses. Our machine is equipped to detect up to 17 colours simultaneously from 4 lasers.

Cell sorting

Cell (particle) sorting can be performed on our FACS Aria II machine. We can sort on to slides, into plates or up to 4-ways into tubes. We can do both simple enrichment sorts to purify your cell of interest, or complex sorts which require multicolour staining to identify and then enrich unique or rare cell populations from a complex sample, such as digested tissue. 

 

 

BD FACS Calibur


call-to-action-science-book-now

2 Lasers provides upto 4 colour analysis

Laser

Detector

Common Fluorophores

Blue laser

(488nm)

FL1: 530/30 FITC, Alexa Fluor488, CFSE, GFP, Cell tracker Green, FDA
FL2: 585/42 PE

FL3: >670

PerCp, PI, 7AAD
Red laser (635nm)

FL4: 661/16

APC, Alexa Fluor647

Example applications:

1-4 colour experiments: Simple immunophenotyping

  • Yeast – live/dead assays
  • Bacteria – live/dead assays
  • eGFP detection – transduction efficiency
  • Cell tracker dyes – cell proliferation

 

BD Arruri C6: 2 Laser/4colour analyser, 2 laser configurations


call-to-action-science-book-now

Configuration one: 3-blue, 1-red

 

Laser Detector Filter Common Fluorophores
Blue Laser (488 nm) FL1 533/30 FITC, Alexa Fluor488, CFSE, GFP, Cell tracker Green FDA
FL2 585/40 PE
FL3 670/LP PerCP, Cy5, PerCP-Cy5.5, Cy7, PI, 7-AAD
FL3 610/20 RFP, PI, PE-CF594
Red Laser (640 nm) FL4 675/25 APC, Alexa Fluor647
       

Configeration two: 2-blue, 2-red

 

Laser Detector Filter Common Fluorophores

Blue laser

(488 nm)

FL1 533/30 FITC. Alexa Fluor488, CFSE, GFP, Cell tracker Green.
FL2 585/40 PE

Red Laser

(640)

FL3 780/60 APC-H7, APC-Cy7
FL4 675/25 APC, Alexa Fluor647

Example Applications:

  • Accurate cell counting
  • Cytometric bead assays
  • Real-time functional assays, eg calcium flux
  • Automated sampling (24 tube rack or 96 well plate)
  • 1-4 colour experiments:    Simple immunophenotyping

 

BD SORP FACSAria II


call-to-action-science-book-now
  • 4 Lasers provide upto 19 parameter analysis (2 parameters for light scattering and 17 for fluorescence) and 4-way cell sorter
  • Current laser configuration enables the simultaneous detection of up to 17 colours.

 

Detectors Band Pass Dichroic Filter Fluorophores  
Blue Laser (488nm) E 530/30

505LP

FITC Alexa-488 CFSE GFP BB515
D 575/25

550LP

PE

   

 

 
C 610/20

600LP

PE-CF594

ECD

PE-TR

PI

 
B 695/40

635LP

PerCP-Cy5.5

Pe-Cy5

PerCP-710

PerCP

 
A 780/60

755LP

PE-Cy7

 

 

 

 
Red Laser (640nm) C 660/20

 

APC

Alexa-647

 

 

 
B 710/50

685LP

Alexa-700

   

 

 
A 780/60

755LP

APC-Cy7

 

 

 

 
UV Laser (355nm) B

450/50

 

DAPI

LIVE/DEAD blue Hoechst blue Indo-1 (violet)  
A

530/30

 

Indo-1 (Blue)

       
Violet laser (405nm) G

 450/50

 

BV421

CellTrace Violet

e450/V450

Pacific Blue  
F

 510/50

502LP

BV510

V500

LIVE/DEAD Aqua

   
E

585/42

570LP

BV570

Zombie yellow

     
D

 610/20

600LP

BV605

Qdot 605

     
C

 660/20

630LP

Qdot 655

BV650

     
B

710/50

685LP

BV711

       
A

780/60

750LP

BV785

       
Top
441-Crop

Example analysis applications:

  • Multicolour immunophenotyping (up to 16 colours simultaneously)
  • Cell lines, isolated primary cells
  • Single cell digestions of complex tissue (eg, skin, fat, lymph nodes)
  • Functional assays
  • Immune cell activation (T cells, monocytes, Antigen presenting Cells)
  • Cell proliferation
  • Cytokine secretion
  • Receptor regulation
  • Bead based assays
  • Cytokine bead assays (eg BD CBA flex set assays)
  • Cell signalling bead assays

 

Cell sorting parameters:

4-way (5ml tubes, 1.2 ml micro  tubes)

2-way (5ml, 15ml)

Single cell deposition

  • Multi-well plates
  • Slides
  • Chamber slides

Multiple sort settings

  • Yield versus purity
  • Aseptic  
  • Temperature controlled

 

Data storage and analysis

Storage of data generated on either of our machines is the responsibility of the user. You will be required to take your data with you following completion of your run. We are not responsible for ensuring your data is backed up. Further instructions will be provided depending on the machine you will be using.

Data analysis is the responsibility of the investigators. The FACS Calibur has on-board analysis software that can be used when the machine is available (Mac based- CellQuestPro). The machine can be booked for long periods of analysis; however data acquisition takes priority over analysis.

Data generated from the FACS Aria II is the responsibility of the investigator, as a separate software package is required. We use the dongle-run software package FlowJo by TreeStar which is compatible on both PCs and Macs (version X). FlowJo tutorials can be accessed from their website http://www.flowjo.com/tutorials/. Depending on demand we can lend out these dongles. Please contact us for more information.

Use of the instruments:

  • Researchers can be trained to use the machines themselves or pay to have an operator assist.

  • Cell sorting will generally require an experienced operator to assist, although this can also be trained if the researcher will be performing this regularly.

Collaborations:

  • We are always keen for new collaborations – especially multicolour panel development. We are also keen to develop assays and to try new techniques. Please get in touch if this is something that would advance your research.

     

Our people


Please contact Anna Brooks for more information or assistance.

Dr Anna Brooks manages the flow centre and holds a BCA (management) and a PhD in Immunology. She is a research fellow with the Maurice Wilkins Centre at the School of Biological Sciences where the flow cytometry centre is located.  Anna has many years experience in flow cytometry and is skilled in a wide range of flow cytometry techniques, including cell sorting. She particularly enjoys the more advanced area of developing multicolour panels (detection of 14-16 markers simultaneously) for the analysis of cell populations in complex tissues (human blood, skin, lymph nodes, liver, brain).