Cell suicide is not painless for the flow cytometrist: new assays to measure cell death

Stevenage , Hertfordshire
Wednesday, 07 November 2012

The venue for this event will be 

The Stevenage Bioscience Catalyst, within the GSK complex,  Stevenage, UK
The Stevenage Bioscience Catalyst campus is a unique bioscience community created to provide small biotech and life sciences companies and start-ups with access to the expertise, networks and scientific facilities traditionally associated with multinational pharmaceutical companies.

This is a Euroscicon Discussion Forum,  an outline of the day can be found at 


Cell suicide is not painless for the flow cytometrist: new assays to measure cell death
Wednesday, 07 November 2012 09:00 - 17:00

The Stevenage Bioscience Catalyst
Gunnels Wood Road
United Kingdom

Map and Directions
This interactive workshop will discuss numerous new assays and approaches to measure autophagy in living cells by flow cytometry, image flow cytometry and fluorescent microscopy as well as new approaches to the measurement of apoptosis. Autophagic cell death has been implicated in cancer and neurodegenerative disorders resulting in new approaches to the treatment of such diseases. The workshop will include talks with questions & answer sessions on a autophagic gene knockdown approach to the study of autophagy as well as the measurement of the autophagosome microtubule, LC3B and lysosome recruitment during the autophagic process in numerous fields of biology including melanoma, immunology as well as cell senescence.

This meeting is for obviously people studying autophagy and  also researchers in areas such as cancer treatments and neurodegenerative disorders as well as flow cytometrists

Meeting Chair: Dr. Gary Warnes
Blizard Institute, Barts & The London School of Medicine & Dentistry, Queen Mary University
This event has CPD accreditation 

Who should attend:  
Anyone studying Autophagy
Researchers in areas such as cancer treatments and neurodegenerative disorders
Flow cytometrists
Amnis machine users 
Facs machine users 
Confocal Imaging machine users

Structure of the day

9:00 – 9:30          Registration

9:30 – 9:35          Introduction by Meeting Coordinator:   Introduction by Meeting Coordinator: Dr Astrid Englezou , Euroscicon, London, UK

9:35 – 9:45         Introduction by the Chair : Dr. Gary Warnes Blizard Institute, Barts & The London School of Medicine & Dentistry, Queen Mary University
9:45 – 10:15     A new flow ctyometric assay for the study of autophagy
Dr. Gary Warnes,  Blizard Institute, Barts & The London School of  Medicine & Dentistry, Queen Mary University
The flow cytometric use of LysoTracker dyes was employed to investigate the autophagic process, using a range of different treatments to induce autophagy. LysoTracker dyes have been employed in microscopy to image acidic spherical organelles,  their use in flow cytometry has not been extensively investigated. During autophagy the LysoTracker signal increases in a time and dose dependent manner mirroring the increase in microtubule protein, LC3B upregulation. The use of LysoTracker allows, unlike the use of antibody labelling technique, the simultaneous measurement of functional parameters. This method has the advantage of other live cell LCB-GFP tagged experiments in that it is easier to use and significantly less costly. 

10:15 – 10:45 Autophagy in keratinocytes
Dr Daniele Bergamaschi, Barts and The London School of Medicine and Dentistry, London, UK
Autophagy has been shown either as a pro-survival or a cell death pathway depending on cell lineage as well as on environmental conditions. 
Although this phenomenon has been well described in fibroblasts and melanocytes, it has yet has to be fully characterized in the major cellular components of the epidermis such as keratinoytes. 
Recent findings will be reviewed to help understand how this metabolic pathway occurs and how it can be modulated in ketatinocytes.

10:45 – 11:15     A novel method for autophagy detection using Image Stream in primary cells: Impaired levels of macroautophagy in immunosenescent T cells.
Dr Kanchan Phadwal, University of Oxford UK
Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration and cancer; however, to date, conventional autophagy detection techniques are not suitable for clinical samples. We have developed a high throughput, statistically robust technique that quantitates autophagy in primary human leukocytes using the Image stream, an imaging flow cytometer. We validate this method on cell lines and primary cells knocked down for essential autophagy genes. A. Furthermore our results indicate that healthy primary senescent CD8 (+) T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age. This technique will allow us, for the first time, to measure autophagy levels in diseases with a known link to autophagy, while also determining the contribution of autophagy to the efficacy of drugs.

11:15 – 11:45 Mid-morning Break

11:45– 12:15     Tools for studying proliferation and cell death by flow cytometer
Dr Niga Nawroly, eBioscience, UK
This  talk will cover cell proliferation discussing current and new toold to measure cell death and proliferation by flow cytometer

12:15  – 12:45      Flow cytometric assays of apoptosis and cell death
Dr Michael G Ormerod, Consultant, UK
Flow cytometry should not be the primary tool to determine whether cell death is occurring through apoptosis. Where possible this should be achieved by observing the nuclear morphology. Flow cytometry is usually the preferred technology for counting apoptotic cells. The choice of assay will depend on the properties of cell being studied. For routine assys, a count of apoptotic cells may be unnecessary; a simple assay to measure cell death (that is, loss of membrane integrity) may be sufficient.

12:45 -13:15     Working Lunch 

13:15  – 14:30   Discussion Group Sessions 1 - 3

14:30 – 15:00    Case Study: Cell Death Methods for Characterisation of a Monoclonal Antibody
Dr Sarah Jones, BioOutsource, UK
Whilst frequently employed as a method by which to detect cell death, the use of flow cytometry is can prove difficult within a  GMP environment. This talk describes several GMP-compliant assays to measure cell death in response to various clinically relevant treatments, focussing on the anti-TNFa monoclonal antibody Humira (adalimumab). Assays such as antibody- and complement- dependent cytotoxicity, coupled with technology from Meso Scale Discovery to measure multiple apoptosis markers simultaneously, mark a leap forward in GMP-compliant measurement of cell death.

15:00 – 15:45       Discussion Group Sessions 4 - 5

15:45  – 16:20      Question and Answer Session
This session will include summing up of the discussion tables and questions submitted both prior to the meeting and throughout the day

16:20 – 16:30         Chairman’s Summing Up and Feedback Prize Draw

Discussion tables
Table A: Dr. Gary Warnes,  Blizard Institute, Barts & The London School of  Medicine & Dentistry, Queen Mary University
Table B: Dr Daniele Bergamaschi, Barts and The London School of Medicine and Dentistry, London, UK
Table C: Dr Kanchan Phadwal, University of Oxford
Table D: Niga Nawroly, eBioscience, UK
Table E: Dr Michael G Ormerod, Consultant, UK

Keywords:  Autophagy, Necrobiology, Apoptosis, Flow cytometry, apoptosis, Image Stream, T cells, Immunosenescence,  Wnt, colorectal cancer, GMP-compliant, cytotoxicity Humira, ADCC, CDC, Cell death, proliferation dyes, 

About the chair
Gary Warnes interest in flow cytometry started at St. Mary’s in 1986, analyzing T-cell subsets. Then set up a new flow cytometric T-cell subset service at St.Thomas’ Hospital. Completed a PhD investigating the immunosuppression of HIV-ve haemophiliacs at St.Thomas’ Hospital. Post-doctoral position, investigated the regulation of Tissue Factor expression by immune co-stimulatory molecules in sepsis. Then managed the Flow & Imaging Core Facilities at the MRC Clinical Science Centre at Hammersmith Hospital. Worked with Derek Davies at Cancer Research UK. Currently managing the Flow facility at the Blizard Institute, Queen Mary University
About the Speakers
Michael G Ormerod  was previously employed as a Senior Scientist Scientist at the Institute of Cancer Research. London. Since taking early retirement, he has been self-employed as research Consultant and trainer. He has taught on courses on flow cytometry in venues, world-side. He recently pblished 'Flow Ctometry - a Basic Introduction, avaliable at http://flowbook.denovosoftware.com/Flow_Book .

Sarah Jones recently joined BioOutsource as a Study Manager - BioAnalytical from her previous role as post-doctoral researcher. After gaining a PhD in inflammatory intracellular signalling, Sarah spent time as an academic researcher in the field of vaccine development before joining BioOutsource. Building on her previous experience, Sarah is interested in the use of Bioassays to support the Biosimilars Industry.

Niga Nawroly was Core lab manager- 10 years Imperial College and is now the secretary of the London flow Club, Honorary Sec of RMS (cytometry section), Application scientisit in eBioscicnce

Dr Kanchan Phadwal is investigating whether immunosenescence (especially T cells) in humans is due to falling autophagy levels, with a long-term aim to manipulate autophagy to rejuvenate the immune system. He is designing in vitro experiments for long-term T cell cultures where autophagy could be either modulated genetically or pharmacologically in order to revive T cellaging.  His  research is also focused towards an in-depth study of DNA damage (DNA double-strand breaks) at telomeres ends in cells with impaired levels of autophagy with a goal to understand their role in contribution to genome instability leading to tumorigenesis.
Since September 2009, he has been involved in the development of a novel assay to detect autophagic flux in primary cells; this is based on colocalisation of autophagosomal and lysosomal markers using a new generation imaging flow cytometer ‘Image Stream100 and X’.   


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