The Dame Charmian O'Connor Faculty of Science - Leading creativity and innovation in the sciences


Auckland Cytometry


Auckland Cytometry is a flow cytometry core facility which provides researchers with technical expertise, training and access to state-of-the-art technologies and applications in flow cytometry.

We can support your research by offering access to:

  • training to use the analysers
  • trained staff to assist with analysis or cell sorting
  • consultation or collaborations to assist with experimental design and data analyses

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.

 

Analysers


BD Accuri C6 flow cytometer

2 laser/4 colour analyser

Configurations: 3-blue, 1-red or 2-blue, 2-red

Configuration one: 3-blue, 1-red

Laser

Detector

Filter

Common Fluorophores

Blue laser

(488nm)

FL1

533/30

FITC, Alexa Fluor488, CFSE, GFP, Cell tracker Green, FDA

FL2

585/40

PE

FL3

670 LP

PerCP, PE-Cy5, PerCP-Cy5.5, PE-Cy7, PI, 7-AAD

610/20

RFP, PI, PE-CF594

Red laser (640nm)

FL4

675/25

APC, Alexa Fluor647

Configuration two: 2-blue, 2-red

 

Laser

Detector

Detector

Common Fluorophores

Blue laser

(488nm)

FL1

533/30

FITC, Alexa Fluor488, CFSE, GFP, Cell tracker Green, FDA

FL2

585/40

PE

Red laser (640nm)

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 (unique to this machine)
  • Automated sampling (24 tube rack or 96 well plate, unique to this machine)
  • 1-4 colour experiments:
    • Simple immunophenotyping

Spectral Analysers

Spectral cytometry is an exciting new technology we are proud to host at the facility. As opposed to conventional detection, spectral cytometers lack individual detectors for the measurement of a dedicated fluorophore. Rather, a “spectral signature” is registered for each particle or cell that is interrogated, and this spectral signature is deconvoluted in order to extract intensity data for each dye. This means that we can use many more dyes in combinations not possible on conventional instruments.

Cytek Northern Lights spectral analyser (available soon):

2 laser (blue/red)

24 channels (at least 10 colours)

Specs for this instrument will be added soon!

Cytek Aurora spectral analyser (coming soon):

4 lasers (blue/red/yellow-green/violet)

50 channels (at least 24 colours). Includes a high throughput auto-sampler

Specs for this instrument will be added soon!

Example applications on spectral analysers

  • Volumetric particle (cell) counting
  • Fluorescent protein detection (eg eGFP) – transduction efficiency
  • Functional assays (ie live/dead /cell proliferation, cytokine secretion, receptor regulation)
  • Multicolour phenotyping (at least 24 colours on the Cytek Aurora)
  • Autofluorescent cell studies -primary tissue digests/cultured cell lines (Cytek instruments)
  • Microvesicle studies (Cytek Aurora)

 

Top

Cell sorter


BD SORP FACS Aria II cell sorter

4 lasers (blue/red/UV/violet)

17 colour analyser 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

BB700

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

APC/

Fire750 

 

 

 

UV Laser (355nm)

C

450/50

 

C

DAPI

 

 

 

B

530/30

505LP

B

BUV395

 

 

 

A

780/60

635LP

A

BUV737

BUV805

 

 

Violet laser (405nm)

 

 

F

 450/50

 

BV421

CellTrace Violet

SB436

 

 

E

 510/50

502LP

BV510

BV480

 

 

 

D

 610/20

600LP

BV605

SB600

 

 

 

C

 660/20

630LP

BV650

SB645

 

 

 

B

710/50

685LP

BV711

SB702

 

 

 

A

780/60

750LP

BV785

 BV786

 SB780

 

 

Example applications

  • Multicolour Immunophenotyping (up to 16 colours simultaneously)
    • Cell lines, isolated primary cells
    • Single cell digestions of complex tissue (eg, skin, fat, lymph nodes)
  • Single cell deposition (sorting) –cloning from single cells
  • Identification and isolation of transduced (fluorescent) cells
    • Molecular characterisation/functional assays
  • Enriching cells of interest/depleting cells/debris

 

Bead assay reader


MAGPIX

Highly specialized magnetic-based system that reads multiplexed bead assays in biological samples

Top

Genomic Cytometry techniques


Single cell RNAseq

In collaboration with Auckland Genomics, we can offer access to:

  • Consultation or collaborations to assist with cell preparation for single cell applications
    • Cell sorting/viability assessment/sample “clean-up” (Auckland Cytometry)
    • CITEseq/Hash tagging (Auckland Cytometry)
    • 10x Chromium Single Cell 3’ Libraries preparation and Next Generation Sequencing (Auckland Genomics)
Top

Data Storage and Analysis


Storage of data generated on any 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. Google Drive is an efficient way of transporting your data, as all machines will have internet access.

Data generated from all the instruments is the responsibility of the investigator, as a separate software package is often required. Data analysis is the responsibility of the investigators although training, and assistance can be provided. We use the dongle-run software package FlowJo by TreeStar which is compatible on both PCs and Macs. 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.

Top

How things work


  • 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.
Top

Collaborations


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

Our people


Dr Anna Brooks (Director) is a Research Fellow with the Maurice Wilkins Centre at the School of Biological Sciences. She has been using flow cytometry for most of her research years, but has more recently been managing and enhancing the capabilities of the flow cytometry facility housed in the School of Biological Sciences for the last 8 or so years. Anna is experienced in a wide range of flow cytometry techniques, including cell sorting, and the more advanced area of developing multicolour panels (detection of 17 markers simultaneously) for the analysis of cell populations in complex tissues (human blood, skin, lymph nodes, liver, brain). Anna is also an active member of the international flow cytometry community and currently sits on the Australasian Cytometry Society committee as Research Liaison.

See, Selected Publications

Brad Wackrow (Laboratory Technician) is the newest member of the flow cytometry team and provides technical assistance for all our instrumentation.