Leeds Imaging and Flow Cytometry Day 2012

We held our second flow cytometry technology day at the University of Leeds on Friday 23rd November, this year we extended it to include high resolution imaging this year to reflect the establishment and work being carried out in the new Imaging Facility in LIMM at the St James' Teaching Hospital site.

The day began with an introduction to flow cytometry presented by Dr Beverly Cork of Life Technologies and was followed by Dr Gina Scott from the St James' site on the use of flow cytometry in following NK cell fate and function. Dr Morgan Blaylock of Becton Dickinson presented the new FACSJazz cell sorter, an instrument designed to fit into labs and perform routine sorting tasks. It also fits into a standard Cat II hood to allow sterile sorting to be performed. The morning session was then wrapped up with the first talk incorporating confocal imaging by Ms Abbie Nelson who showed great images of NK cells and the formation of lytic granules acquired on the Nikon A1R confocal.

Dr Bev Cork of Life Technologies presenting an  introduction to flow cytometry in the morning session.

After a buffet lunch kindly provided by our sponsors (who hopefully had lots of interest from the delegates!) we had a session on high resolution imaging from speakers based on main campus with the Faculty of Biological Sciences (FBS). The session however was kicked off by Dr Chris Power from Zeiss who presented an overview of optical microscopy techniques. Additionally he introduced a recently developed technology referred to as light sheet microscopy (SPIM). With this technology fantastic images of developing embryos can be acquired super fast in 3D and is being utilised in the study of gene expression in the developing drosophilla embryo. Subsequent talks covered an array of subject areas from the role of SNARE proteins in NK cell cytotoxicity (Dr Andrew Hellewell, FBS), to prion proteins in Alzheimer's disease (Dr Heledd Griffiths). Prof Jurgen Deneke (School of Biology, FBS) presented the talk of the day on image selection and statistical analysis of colocalization; making a talk on statistics entertaining!!

Prof Jurgen Deneke (School of Biology, FBS, University of Leeds) presenting a talk on image selection and analysis of confocal data.

Perusing the kind sponsor's stalls during the break!

Our final session had presentations on 'new technologies' and covered developments in mass cytometry, high throughput flow cytometry, multiparameter imaging and super resolution microscopy. Dr Jane Limer of DVS Sciences presented a new generation cytometer called CyTOF which combines the single cell analysis of a flow cytometer but extends the potential number of marker detectable to nearly a 100 in a single sample! Wow, amazing stuff!! Dr Erika Wilson (LIMM) presented data that her group have acquired using LyoScreen plates from BD Biosciences to investigate the surface antigens on cancer related immune cells. We were presented with some fantastic images of in vivo angiogenesis by Dr Kathryn McMahon (LIMM) who uses confocal imaging and tiling to acquire large areas of fluorescently labelled mouse retina. Finally, the meeting was wrapped up by Dr Dmitry Ushakov from Prof Michelle Peckham's lab in FBS presented the recent development of PALM and STORM within the Faculty.

Cheese and wine (and chips!) for the end of day delegates!

To finish off I just want to give a huge THANKS to the kind sponsors who made this event such a success. Onward now to 2013..!

Kinetic studis using flow cytometry

One parameter that isn't used to a great extent in our facility, if at all, is the time parameter. From a diagnostic point of view time is useful to assess flow rate; if we do a dot plot with time as the x-parameter and fluorescence for example on the y-axis we can determine the flow and whether or not it is disrupted as the sample goes through.

We can however use it to assess dynamic changes in samples, for example to assess changes in fluorescence if we use a calcium or other ion indicator.

A number of papers have been published on this. We can use dyes such as Fluo3, which is excited by the blue 488 laser and emits in the green FL1 channel (Calibur). As with many dyes of this sort fluorescence intensity increases upon binding of calcium. Ratiometric measurements require a near-UV laser to excite Indo-1 and so is restricted to instruments with this excitation source. Recently however the green dye referred to above can be used in conjunction with Fura Red to perform ratiometric imaging by collecting green and red fluorescence simultaniosly. With the advent of more collection channels and lasers we can now of course combine calcium measurements with immunophenotyping and viability assays to really unpick the complxities of multicellular systems.

One issue that exists however with performing calcium flux on machines that are presurised (eg FACSCalibur) is that sample tubes have to be physically removed from the intake port, agonist added, then placed back on for further acquisition. This produces a gap in the data. With this gap its hard to determine what has occurred during this initial stage. A system from Accuri appears to have overcome this. As it is not a pressurised system one can add agonists or other stimulants directly into the tube as acquisition occurs negating the need to pause acquisition and preventing the loss of data we see on more conventional systems. Neat.

We have recently been contacted by a researcher interested in multicolour calcium imaging and we are currently planning to conduct a workshop this autumn on the subject. If you would be interested please let me know.

Practicalities of Flow Cytometry meeting, Newcastle-Upon-Tyne, Feb 23rd 2010

A quick jaunt up the tracks to Newcastle was well worth it for an excellent meeting on the practicalities of flow cytometry hosted by Ian Dimmick and Becky Stewart of Newcastle University. In a unique blend of commercial and research talks a number of different applications and tools were presented.

The morning began with talks about cytokines secretion capture systems (Miltenyi) and their applications to the study of IFNg secretion from NK cells in Cowdens Disease (Ian Dimmick). We also had a presentation on the cell permeable DNA dyes DRAQ5 and CyTrack Orange (Biostatus) and their applications in studying the cell cycle in live cells and ensuring we look at nucleated cells in peripheral blood samples without having to Lysenko RBCs. Allied with that Prof Paul Smith from Cardiff presented fascinating data on the use of a compound called AQ4N which is activated in hypoxic conditions (when it's converted to AQ4) such as within the microenvironment of tumours where a number of anti-cancer drugs are incapable of eliciting their effect.

On a diversion from mammalian cells we had a presentation on the applications of FCM in the study of plant genome size (Ilia Leitch, Kew). It appears the size of a plant genome may regulate a number of factors such as life cycle: weeds which grow quickly have small genomes while obligate perennials are slower growing and have large genomes. Plants with large genomes are generally not found in areas of extreme ecological stresses. And onions have 5 times as much DNA as we do! Maybe they are more intelligent!?

The post coffee session kicked off with BD's Flex bead assays and their use in the investigation of immunity amongst professional footballers. This was followed by presentation of the fixed alignment cytometer C6 from Accuri (Kate Easten) and it's use in studying calcium fluxes in research (Blanco Fernande, Dublin). A very powerful piece of equipment and so easy to operate. Definitely one for the wish list I think!

The afternoon was dedicated to multicolour flow and data analysis. Becky gave an excellent overview of compensation and we heard a great presentation on the use of 11 colour flow analysis in studying clinical cardiology.

The analysis of multicolour flow data can be very complicated and a number of approaches are available to analyse this type of data. Trevor Ray of Caltag presented a commercial solution in the form of Infinicyt, while Karen Fiser of Newcastle Uni presented a more 'home made' solution based on hierarchial clustering of data.

I missed the last couple of talks as I had to catch my train (!) but all-in-all a great day of flow information and a number of interesting applications were discussed in an inspiring environment.