Flow Cytometry

The Fiona Elsey Cancer Research Institute is one of only a few laboratories that have a dedicated sorting and analysis machine within Australia.

Definitions

  • Flow Cytometry is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus

  • FACS - Fluorescence-activated cell sorting is a specialised type of flow cytometry

Our Flow Cytometer History

The Fiona Elsey Cancer Research Institute is one of only a few laboratories that have a dedicated sorting and analysis machine within Australia.

The flow cytometry facility at the Fiona Elsey Cancer Research Institute (FECRI) was initially funded by the estate of Harry and Peggy Maddicks. This funding was used to purchase a BD FACSCanto in 2007. This machine allowed the researchers to analyse their samples and start to phenotype the different cell markers within LCH lesions. BD Bioscience generously allowed the Institute to upgrade this machine in 2008 to a FACSAria II, which allows for both analysis and cell sorting. This upgrade was funded through generous donations from Stewart and Sue Gull as well as Jim and Val Selkirk. Both families are based in the Ballarat community.

The cell sorting function of this machine is vital for both the LCH and apoptosis projects underway at the centre. BD Bioscience not only facilitated the exchange of the Canto to the Aria but also upgraded the FACSAria II with 3 lasers to allow for 9 colour analysis. They continue to support the flow cytometry facility through regular maintenance of the machine and supply of reagents at reduced costs.

The FACSAria flow cytometer at the Institute is currently operated and maintained by Jenny Luke. Jenny came to the centre in 2008 from the Walter and Eliza Hall Institute flow cytometry facility in Melbourne. An experienced cytometry technician, Jenny has been involved in the setup and teaching of flow cytometry techniques to the staff at the InstituteI.

Uses of the Flow Cytometer at The Institute

  • The FACSAria II is currently being used in the centre for the following research:

  • LCH project - Vital for the phenotyping of LCH cells. FACS is also being used in this project to sort different cells within the LCH lesion in order to fully comprehend the micro environment required for the proliferation of LCH

  • The Biomedical Science program at the Federation University also uses the facility for student practicals in Immunology

Cell Sorting (FACS)

Our flow cytometer is also capable of sorting cells based on the properties measured by fluorescence. The mechanism of cell sorting and analysis begins the same way. A cell suspension is taken from the sample input chamber and injected into the centre of a saline stream. The stream and cells pass through a tapered nozzle after which each cell is illuminated by the laser beams. Data is collected at this point and a decision can be made as to which cells are to be collected.

A high frequency vibration is applied to the nozzle allowing for the stream to be broken up into droplets at a set distance from the tip. This distance is stable and the time elapsed between the nozzle and the break off point can be determined through the cytometry program. Once the break off is determined the machine is able to apply an electrical charge to each drop and then deflect (sort) the droplet that contains the cells of interest into a specific sort tube.

The cells collected are essentially unharmed by the process and thus can be used subsequently for in vitro or in vivo responses or analyzed at the RNA or DNA level.

The cells may be sorted into tubes where large numbers are required or small, accurately counted numbers or even individual cells may be deposited directly into microtitre culture plates.

References