Cell-sorting / Flow Cytometry
Flow cytometry is a powerful tool for the multiparameter analysis of all types of cells. It analyses cells or particles as they move in a liquid stream through a laser or light beam past a sensing area. Analysis and differentiation of the cells is based on size and granularity and whether the cell is carrying fluorescent molecules either in the form of antibodies or dyes.
The flow cytometer consists of a light source, collection optics, electronics and a computer to translate signals to data. The light source of choice is a laser which emits coherent light at a specified wavelength. Scattered and emitted fluorescent light is collected by two lenses, one set in front of the light source (forward scatter) and one set at right angles (side scatter) and by a series of optics, beam splitters and filters, specific bands of fluorescence can be measured.
An example of a cell death assay using AnnexinV and Propidium iodide to identify apoptotic cells with Flow Cytometry is shown below:
The Figure above illustrates an experiment to assess cell death using AnnexinV (FITC conjugated) and PI in MCF7 cells. The top panel are control cells and the in the one below cells were treated with TRAIL 200ng/ml for 4h (Gareth Miles, 2011).
IN THE HODGKIN BUILDING WE HAVE FOUR FLOW CYTOMETERS AVAILABE.
The analysers Calibur and FACS Canto II are located in room 424 and the FACScan in room 310. At the moment, the bookings are done in the diaries placed nearby or the board located next to the machines. Any new user, please see Dr Lucia Piñon (Ext 5569) for advice and instructions.
The FACS sorter Aria II is located in room 419 and use/ bookings are exclusively through Dr Lucia Piñón. Please see booking form.
The BD FACS Canto II is an analyser with two lasers,a blue argon laser (4 colours) and a red HeNe (2 colours) and thus capable of detecting 6 fluorescent signals e.g. FITC, PE, PE-Cy5.5, PE-Cy7, APC and APC-Cy7. The software used is Diva 6.1.2 and after each experiment the data should be exported to D: BDShared and removed from the Browser.
The BD FACS Calibur is used for analysis only. It has two lasers. The primary laser is an air-cooled 15mW argon ion laser emitting at 488nm, thus allowing two light scatter parameters and three fluorescence channels to be measured. The second laser is a red diode laser emitting 635nm thus allowing the excitation of other dyes such as allophycocyanin or TO-PRO-3. The software present is CellQuestPro v6.0
The FACScan is an analyser with one argon laser capable of detecting 3 fluorescent signals e.g. FITC or GFP, PE and PE-Cy5 or PE-Cy7 and the software for acquisition and analysis is CellQuestPro v.6.0
The BD FACSAria II is an analyser and cell sorter with five lasers instrument and has an aerosol management installed. It can detect 16 colours simultaneously.
The FACSAria can perform 2 and 4 way sorting as well as automatic cell deposition for sorting onto TC plates or slides. Sort rates are 20,000 cells per second for lymphocytes and 10,000 cells per second for cells >10um via 70, 85 and 100um nozzle sizes at 70 and 20 psi respectively. Additional to the standard Blue (488 nm), Yellow Green (561 nm) and Red (640 nm) lasers, there are also UV (355 nm) and Violet (405 nm) lasers available.
If you are planning to use the ARIAII sorter it is recommended to discuss your choice of dyes before ordering the antibodies/compounds to ensure they fit the instrument configurations.
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 ). A minimum concentration of 1 million cells per ml and a minimum volume of 500 ul are recommended for sorting and analysis. For the first set-up of any experiment a sample with unlabelled cells - and for multicolour experiments additional samples with single stains (one colour only) - are required.
There is a PC computer available in room 423 for pure data analysis with FlowJo software for general FACS applications. The D:BDShare folder from Canto and ARIAII and the Calibur are available on this computer.
All copyrights in this website, including logos, illustrations, photos, sound, scripts, animation and other material appearing on this website, otherwise known as "content", belong to the MRC Toxicology Unit or third parties authorising the MRC to use the copyrights.
Users must not reprint or electronically reproduce from the MRC Toxicology Unit website any graphic in whole or in part without the prior written permission of the MRC Toxicology Unit.
Users may download and save electronic documents, audio files and forms from the Website and print or listen to these for personal use, but such forms, files and documents remain the copyright of the MRC Toxicology Unit, 2013.
MRC Toxicology Unit
PO Box 138
Lancaster Road, Leicester
LE1 9HN, UK
SatNav Users: Using this postcode will not direct you to the correct location. Please search for Lancaster Road.
Tel: +44 (0)116 252 5544
Fax: +44 (0)116 252 5616