FACS Aria II Flow Cytometer

 

Introduction

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 :

Configurations

The FACSAria II of the department "Cancer Studies and Molecular Medicine"  (CSMM) 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). For example the blue Laser can only be used with dyes which are excited at 488 nm and emit at 530 nm such as FITC (Fluorescein isothiocyanate) or dyes which excite at 488 nm and emit at 710 nm like PerCP. 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. Under exceptional circumstances it is, however, also possible to change the configuration for a laser if the required filter is available. The following link will help you with your selection of dyes:

Sample format

Your cells have to be in a liquid suspension either in 15 ml Falcon tubes (BD Falcon, Cat# 352196), 5 ml FACS tubes - preferred (12 x 75 mm, BD Falcon Cat# 352054 ) or 0.5 ml 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 a sample with unlabelled cells - and for multicolour experiments additional samples with single stains (one colour only) - are required. Fluorescence minus one (FMO) controls are also suggested to check for spectral overlap in multicolour experiments.

Sorting

FACSAria II is specifically designed to sort cells at high flow speeds.  The sorted cells are collected in a variety of vessels including: 15 ml Falcon tubes for 2-way sort, 5 ml FACS tubes (Polypropylene, BD Falcon Cat#352063) for 4-way sort, 1.5 ml and 1.0 ml microcentrifuge tubes, 6-,12-,48- and 96-well plates and 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. Multi-way sorting is possible according to the dyes and collection vessels used. The sorts can be sterile or non-sterile and will be set up by the FACS operator according to your requirements.

Data Analysis

FACSDiva software Version 6.1.2 (BD Biocsiences, San Jose, USA) is installed on the FACSAriaII for operating the instrument and analysing the measurements.

There is a second computer (Network Computer) available in the FACS room for pure data analysis. It runs the FACSDiva and WinMDI software for general FACS applications as well as the MODFIT program for Cell Cycle analysis. This computer is connected to the University network, where you can store your analysed data in your personal folder (z-drive). You can import your data from the archive folder into FACSDiva by using the import function in the file menu.  A shortcut to the archive folder will be sent to you by the FACS operator upon request after your name has been added to the user lists for the archive and the FACSDiva program. No booking is required for the Network computer.

For a video tutorial on data analysis, multicolour experiments and compensation, please click the link below:

Users

Everybody is welcome to use the CSMM FACSAriaII, but CSMM users will have priority. 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.

User Form

FACSform (pdf)                            FACSform (Word)

 

Costs

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. 

Booking

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.

There is an electronic booking system in place to book time slots on the FACS. Booking is only possible with Microsoft Outlook Calendar and from a University of Leicester network computer due to technical reasons. Please see instructions below on how to access this calandar:

  • Open Microsoft outlook 2010
  • Click Calendar button
  • Click on “Open calendar”
  • From the drop down menu choose "From Address book"
  • Type “CSMM” into search field
  • Highlight “CSMM_FACS1_Booking”
  • Press calendar button and press ok.
  • FACS calendar will appear next to your personal calendar
  • Make sure you have not chosen the overlay option in the view tab to make sure the calendars appear side by side.

If you do not have access to an University computer you can arrange with the FACS operator to do the booking for you.

Data aquistion bookings must include;

  • Your Name
  • Stipulate aquistion only

Sort bookings must include;

  • Your Name
  • 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 patient samples) 

Please add 30 min for the setup and sterilisation of the instrument prior to your booking, indicatated as SORT SET-UP

Bookings can only be changed or cancelled by the FACS operator. Changes made 2 hours prior to your booking will not be charged, bookings changed after this time will still be charged for the time booked and any subsequent time used. 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.

Data transfer

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.

It is recommended to include the date in the experiment name, since transfer date and acquisition date might be different.

If you are a user without a University of Leicester account, you can download your data through a password protected FTP account. You need to install WinSCP  or another FTP exchange programm on your computer and will receive instructions from the FACS operator how to log into this FTP account.

Please note:  Personal USB Memory sticks are not allowed to be used on the FACS Computer due to the risk of virus infections. 

General Rules

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.

Location

The FACSAria II is located in Room 515A, Level 5, Robert Kilpatrick Clinical Science Building and belongs to the group of Dr. Karen Brown (kb20@le.ac.uk).

Operator

Dr Jennifer Higgins

jah52@le.ac.uk

Cancer Studies and Molecular Medicine
Chemoprevention and Biomarker Group (Dr Karen Brown)
Robert Kilpatrick Clinical Science Building, level 5,

Royal Infirmary
Leicester
LE1 5WW

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Contact Details

Department of Cancer Studies & Molecular Medicine
Clinical Sciences Building
University of Leicester
Leicester Royal Infirmary
Leicester LE2 7LX

T: +44 (0) 116 258 7597
F: +44 (0) 116 258 7599
E: csmm@leicester.ac.uk