FACS Aria II Flow Cytometer
The FACSAria II from Becton Dickinson (BD Biocsiences, San Jose, USA) is a high speed cell sorter for measuring and sorting fluorescently labelled cells. These cells can be prokaryotic (e.g. bacteria) or eukaryotic (e.g. mammalian) with a minimum size of 0.2 um (maximum 100 um). Fluorescence staining is usually performed with antibodies specific to the proteins of interest, which can be intra-cellular or on the cell surface. These primary antibodies are conjugated to a fluorescent dye, or a fluorescent secondary antibody may be used. Fluorescent substrates or compounds can also be detected.
A Laser excites the fluorescent dyes bound to the cells. The emitted light has a longer wavelength and passes through a bandpass filter to shut out the excitatory and other unwanted light. The strength of this fluorescent light is measured with a detector (PMT) for each cell and is displayed in form of a histogram. For more information about the principles of Flow Cytometry, please find below a link to a video tutorial from Invitrogen :
The FACSAria II of the department of Cancer Studies is a customised instrument and has 5 lasers: UV (355 nm), Violet (405 nm), Blue (488 nm), Yellow/Green (561 nm) and Red (640 nm). The FACSAria II has configurations that assign a set of filters to each laser. Which filters are available for what lasers can be found in the User Form (see User Form below). If you are planning to use the CSMM flow cytometer it is recommended to discuss your choice of dyes before ordering the antibodies/compounds to ensure they fit the instrument configurations and to ensure dyes do not overlap resulting in high levels of compensation being required. The following link will help you with your selection of dyes:
Your cells have to be in a liquid suspension either in 15 ml Falcon tubes (BD Falcon, Cat# 352196), 5 ml FACS tubes -(12 x 75 mm, BD Falcon Cat# 352054 ) or PCR tubes. A minimum concentration of 1 million cells per ml and a minimum volume of 500 ul are recommended for sorting and acquisition.
For the first set-up of a new experiment it is essential that a sample with unlabelled cells - and for multicolour experiments, additional samples with single stains (one colour only) - are included. Fluorescence minus one (FMO) controls are also suggested to check for spectral overlap in multicolour experiments.
FACSAria II is specifically designed to sort cells at high flow speeds. The sorted cells are collected in a variety of vessels the most popular are: 15 ml Falcon tubes for 2-way sort, and 5 ml FACS tubes (Polypropylene, BD Falcon Cat#352063) for 4-way sort, it is also posssible to sort into 1.5 ml and 1.0 ml microcentrifuge tubes, 6-,12-,48- and 96-well plates and onto microscope slides. These collection vessels should contain media or buffer and can be cooled to 4 C with a thermostat. Polypropylene FACS tubes are recommended for collection, because they show less cell adhesion than polystyrene ones. The sorts can be sterile or non-sterile and will be set up by the FACS operator according to your requirements.
FACSDiva software Version 6.1.2 (BD Biocsiences, San Jose, USA) is installed on the FACSAriaII for operating the instrument and analysing the measurements.
MODFIT, for Cell Cycle analysis, is available on a separate computer (Network Computer) available in the FACS room for pure data analysis.
For a video tutorial on data analysis, multicolour experiments and compensation, please click the link below:
Before you can use the FACS you will need to fill in the user form (see User Form below) which should be returned to the FACS Operator. On the form you will also be asked to kindly acknowledge the MRC grant, which has funded the FACSAria II, in your publications. This will enable us to demonstrate the various translational applications of the instrument when reporting back to the MRC.
To maintain the high level of service provided we now need to introduce charges for the use of the FACSAia II, in line with the policy of other university facilities. These will be discussed with you when you meet with the FACS Operator to discuss your project.
The primary application of the FACSAria II is sorting. If there are slots available, it can also be booked for data acquisition (just measurements), but sorting will always have priority.
Booking is done via a diary in the FACS room, however, to ensure the machine is available and correctly calibrated for your specific experiement please also email the FACS operator with your booking requirements.
Data aquistion bookings must include;
- Your Name
- Contact details (telephone/email)
- Lasers required
- Experiment description, Stipulating aquistion only
Sort bookings must include;
- Your Name
- Contact details (telephone/email)
- Lasers required
- Experiment description including:
- Temperature of sort (RT or 4ºC),
- Sterile or Non-sterile
- 4-way or 2-way sort
- Collection vessel (Falcon tube, FACS tube, Eppendorf)
- If aerosol is required (primary samples)
Please add 30 min for the setup and sterilisation of the instrument prior to your booking
Bookings can only be changed or cancelled by the FACS operator.
Cancellations to acquisition bookings can be made up to 2 hours prior to booking, excluding those requiring the help of the FACS operator. All bookings requiring the FACS operator or involving sorting must be made 24 hours before your booking. Cancellations made after these times may still be charged at the discretion of the FACS operator. In all instances of changes or cancellations an email must be sent to the FACS operator.
The FACS operator reserves the right to amend or change bookings according to the needs of the department.
Your data will be stored in a secure archive folder in the university network, which is periodically backed up. You will be able to access this data with a network computer after being accepted as a user. The data will be stored in FACSDiva format unless a different format (FCS) has been requested (in the log book).
It is recommended to include the date in the experiment name, since transfer date and acquisition date might be different.
Please note: Personal USB Memory sticks are not allowed to be used on the FACS Computer due to the risk of virus infections.
Eating and drinking are not allowed and protective clothing (lab coats, gloves etc.) has to be worn in the FACS room. If you work with primary cancer cells, lab coat, gloves, face mask, safety glasses and the application of the aerosol managment are required.
If you encounter any problems with the machine immediately contact the FACS operator. After each session the FACS has to be cleaned according to the operators instructions (5 min FACSclean, 5 min deionised water) and the log book has to be filled in. If you do not sign the log book, your data will not be transfered. Do not leave anything in the FACS room or it may be thrown away.
The FACSAria II is located in Room 515A, Level 5, Robert Kilpatrick Clinical Science Building and belongs to the group of Dr. Karen Brown (firstname.lastname@example.org).
Dr Jennifer Higgins
Cancer Studies and Molecular Medicine
Chemoprevention and Biomarker Group (Dr Karen Brown)
Robert Kilpatrick Clinical Science Building, level 5,