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.
For information about the principles of Flow Cytometry, please follow the link below to a video tutorial from Invitrogen :
The Cancer Studies FACSAria II 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 specific configuration currently in use is detailed on the FACS 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 configuration 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:
Please bring your cells in suspension at a minimum concentration of 1 million cells per ml and a minimum volume of 250 ul. Samples can be brought in:
- 15 ml Falcon tubes (BD Falcon, Cat# 352196),
- 5 ml FACS tubes (12 x 75 mm, BD Falcon Cat# 352054 )
- PCR tubes.
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.
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 6.1.3 (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 which should be returned to the FACS Operator. Once you have done this the FACS Operator will contact you and let you know if your experiments can be accommodated and arrange for a mutually convenient time to meet to discuss them further and have an induction.
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.
Charges are in place for use of the FACS. These will be discussed with you when you meet with the FACS Operator to discuss your project. A signed costing agreement must be returned before you may use the FACS.
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.
How to book will be discussed with you when you have your FACS induction.
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, 10 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,