Core Facilities - BD FACSAriaII

Location: Flow Cytometry Facility, BB2-2044, Second Floor South, Bioscience Building.
Person responsible: Eoin Clancy
Email: eoin.clancy@nuigalway.ie
Phone:
 

AriaII charges 2017: €100 start-up charge + €60 per 1 hour.

 

Booking: All sorts must be pre-approved by Eoin Clancy and the yellow sections of the sort booking form completed, the green sections will be completed by Shirley Hanley during the sort. The completed forms will be kept on record in the flow cytometry facility. Please download Sort Booking Form and email to eoin.clancy@nuigalway.ie. The sort booking form must be sent to Eoin Clancy a minimum 24 hours before the start of the sort.

 

A minimum cancellation period of 24 hours applies, sorts cancelled after 24 hours will still be charged.

 

Hours of booking: The AriaII is available for sorts during normal working hours, from Tuesday to Friday, 11am to 5.30pm. The set-up and sorting is performed by the core facility staff only. Out of hours use of the AriaII is not permitted.



 

Flow cytometry is a qualitative and quantitative analysis technology that can characterise cell populations at a single cell level. Flow cytometry is a multi-parametric technology that measures fluorescent signals from the scattered light produced by cells as they pass or 'flow' through a laser light source. These signals can becorrelated to structural and/or functional cell parameters.

Staining cells with fluorescently-labeled antibodies or cell surface-binding molecules allows the identification of subpopulations within a heterogeneous mixture. Subpopulations of cells thus identified can be isolated, or sorted, from the mixture at speeds of approximately 40,000 cells per second and with a purity approaching 100%. This technique is called fluorescence-activated cell sorting.

 

The NCBES flow cytometry core facility has a high speed state of the art BD FACS Aria II cells sorter. The FACS Aria II is capable of detecting up to 11 fluorescent parameters and 2 physical parameter per sample. The AriaII can sort up to 40,000 cells per second and simultaneously sort 4 individual subpopulations with a purity of > 95%. Cells can be sorted into 15ml tubes (2 way sorts only); 5 ml tubes ( 4 way sorts) or  into 24, 48 and 96 well cell culture plates and as well as directly onto microscopic slides.
Sorts are carried out under aseptic conditions, therefore sorted cell populations can be cultured and used for further analysis, or used directly for qPCR analysis.

 

The BD FACS Aria II has 4 lasers and the configuration is as follows:











































































Filter



Primary Flourochrome



Other Flourochromes/dyes



Blue Laser, 488nm



530/30nm



FITC



AF488, GFP, CFSE



585/42nm



PE



PI, RFP



616/23nm



PE-Texas Red



PE-CF594



695/40nm



PerCP



PerCP Cy5.5, PE-C5.5, PI



780/60nm



PE-Cy7











Red Laser, 633nm



660/20nm



APC



AF647



780/60nm



APC-Cy7



APC-H7









Violet Laser, 405nm / Near UV Laser 375nm (both lasers share the same optical path)



450/40nm



Pacific Blue



Cascade Blue, DAPI, Hoechst, AF405, V450



530/30nm



AmCyan



V500




Maximum number of colours that can be detected simultaneously: 9

 

Sample preparation for sorting:

 

Sort samples MUST be in a single cell suspension and resuspended in a sort buffer.


 


FACS SortingBuffer (500ml)


Gibco cat# 14190-094 x1 PhosphateBuffered Saline (Ca/Mg++ free)      480.5 ml


0.5M EDTA (2mM finalconc.)                                                                      2.0 ml


Sigmacat # H0887 1M HEPES pH 7.0  (25mM final conc.)                           12.5ml


FCS (1% final conc)                                                                                     5.0 ml


 Filtersterilize, aliqout and store a 4°C


 



Further information on sort buffers can be found here: 



 http://facs.scripps.edu/SortLink/buffer.htm




 

Information on sample preparation can be found here:

http://www.grc.nia.nih.gov/branches/lmg/fcl/guidelines_sort_sample_prep.htm