University of Southampton

The Cancer Sciences Division houses five Becton Dickinson flow cytometers, a FACScan, a FACSCalibur and a FACSVantage located within the Tenovus building, and a FACSCalibur and a FACSCanto located in the Somers building.  These Divisional facilities are additional to those provided by The Faculty of Medicine Flow Cytometry Facility

 

 

The FACScan has a single 488 nm laser for 3 colour analysis 

 

The FACSCalibur in Tenovus has 488nm  and 633 nm lasers for up to 4 colour analysis, and is routinely used for detection of and APC fluorescence

 

The FACSCalibur in the Somers Building has a single 488 nm laser, and provides basic cell sorting capability

 

The FACSCanto in the Somers building is a  powerful benchtop machine with 488nm and 633 nm lasers allowing six-colour analysis. 

 

These 4 analytical instruments play an essential role in research projects over the whole Division and throughout the Faculty of Medicine.  

 

Applications include:

  

Research students and new staff are given basic training in the use of these machines during their induction course. 

 

The FACSVantage is a cell sorter with 488nm, 633 nm and UV lasers giving 4 excitation wavelengths. From these three laser lines up to five colour analysis can be performed. 

We have considerable expertise in a range of sorting applications including:

In addition to sorting, the Vantage is also used to determine intracellular calcium fluxes (Figure 3)(Du, Honeychurch et al. 2004)

 

Unlike the FACScan and the FACSCalibur, the Vantage cannot be set up and operated by individual researchers. Within the Division, Ruth French (rrf@soton.ac.uk, Tenovus) and Ian Mockridge (cim@soton.ac.uk HIT group) are trained operators. These individuals should be contacted if access to this machine is needed.

An important factor to consider when planning cell isolation is whether they can be collected in a reasonable time. From experience, we have found that people are often disappointed if they want to sort cell for functional analysis. On the basis of total cells running through at 5000 / second, that is 1.8 x 10⁷ /hour. With a yield of, for example 10% of the total, that is a maximum of 1.8 x 10⁶, and realistically 1 x 10⁶ / hour. Pre-enrichment of the required population, for example using MACS beads, may be required. Contact Ruth or Ian to discuss specific requirements..

If you need to obtain a higher sort rate, the Division of Infection, Inflammation and Repair has a FACSAria. Contact Richard Jewell (rj@soton.ac.uk).

 

Tenovus Ext 6590rrf@soton.ac.uk
HIT Group Ian Mockridge Ext 8512 cim@soton.ac.uk
Nasia Kontouli  Ext 6670 A.Kontouli@soton.ac.uk

 

 

Figure 1:
Six colour phenotyping of human blood for the discrimination of T cell memory and functional polarisation using the FACSCanto. Helper T cells are identified through CD3 PerCP and CD4 APC-Cy7. Naïve CDT cells are identified by the markers CCR7 and CD45Ra. Further T cell memory population subdivision in to central memory and effector memory is undertaken by loss of CD45Ra positivity for Central Memory cells and CCR7 for Effector Memory cells. The chemokine receptors CCR4 and CD183 are preferentially expressed on Th2 and Th1 cells respectively. A differential expression of these markers is seen on naïve compared to developing memory populations with polarisation towards pre Th1/Th2 (CM) and committed Th1/Th2 (EM) subsets. Non CD4 cells, predominantly CD8 cells, contrast with CD4 cell by expressing the CD183 whilst naïve and displaying less CCR4 expression upon differentiation 

 

 

Figure 2: 
Purification of a low frequency cell population using the FACSVantage. CD3^-7⁺show TCRγ-chain rearrangement. Flow cytometric analysis of fetal LPLs using CD7-FITC and CD3-PE Abs before sorting (A) and after two rounds of scoring (B) reaching a purity of >99%. C Rearranged TCR-γ chains are detectable within the CD3^-7⁺ cell subset and the CD3⁺ cell subset of fetal gut samples and adult gut samples in the VγJP reaction. PCR products are generated in the size range 70-110 bp and the rearrangement appears to be polyclonal in both subsets, generating bands in the CD3⁺7^- subset and a broad smear in the CD3⁺ subsets

 

 

Figure 3:
Measurement of intracellular calcium flux using the FACSVantage  Intracellular calcium flux in Daudi cells in the presence of ionomycin (positive control),anti-IgM and anti-CD20 was measured after loading of cells with indo-1-AM which shows a shift in fluorescence emission from 482 nm (FL4) to 398 nm (FL5) on binding calcium.

 

Cragg, M. S., S. M. Morgan, et al. (2003). "Complement-mediated lysis by anti-CD20 mAb correlates with segregation into lipid rafts." Blood 101(3):1045-52.

Du, Y., J. Honeychurch, et al. (2004). "Antibody-induced intracellular signaling works in combination with radiation to eradicate lymphoma in radioimmunotherapy." Blood 103(4): 1485-94.

Forconi, F., S. S. Sahota, et al. (2001). "Tumor cells of hairy cell leukemia express multiple clonally related immunoglobulin isotypes via RNA splicing." Blood 98(4): 1174-81.

French, R. R., V. Y. Taraban, et al. (2007). "Eradication of lymphoma by CD8 T cells following anti-CD40 monoclonal antibody therapy is critically dependent on CD27 costimulation." Blood 109(11):4810-5.

Gunther, U., J. A. Holloway, et al. (2005). "Phenotypic characterization of CD3-7+ cells in developing human intestine and an analysis of their ability to differentiate into T cells." J Immunol 174(9): 5414-22.