UCL Drug discovery group application note. Author: Dr.

UCL Drug discovery group application note. Author: Dr.

UCL Drug discovery group application note. Author: Dr. Ben Allsop Cell toxicity assays *Data in this presentation are owned by the UCL DDG and are not for unauthorised reproduction* School of Pharmacy 29-39 Brunswick square WC1N 1AX Translational Research office Types of cell death Cell death is a major feature of disease and an important part of developmental biology. Death may occur in response to external injury, internal cues or programmed redundancy. Necrosis (extrinsic injury induced cell death) Cytoplasmic swelling followed by rupture of the plasma membrane.

Death receptor activation (Fas, TRAIL-Receptor etc.) Release of DNA from the nucleus. Mitochondrial swelling and decreased ATP production. Apoptosis (cell suicide) Membrane blebbing and organelle compartmentalisation Cytochrome C binds and activates procaspase-9 (Apoptosome) Eventually followed by necrotic cell death. Measuring cell toxicity Cell Viability CellTiter-glo (Promega) is an endpoint luminescence assay used to determine cell viability. This assay results in cell lysis and a signal proportional to the amount of ATP present in the well. The amount of ATP is directly proportional to the number of cells present. CellTiter-glo is a simple 1-step addition at a fixed timepoint. NB: Luminescence readouts are often O2 dependent and may have a reduced signal in

hypoxia. Cell death CellTox-Green (Promega) is green channel fluorescent assay able to directly measure cell death. This assay uses a non-cell permeable DNA binding flurophore, meaning it can directly measure the free-DNA of burst cells (Necrotic death). CellTox-Green is well tolerated and non-toxic, it can be used kinetically to measure cell death over hours or days. Kinetic measurement of cell death Measuring cell death throughout reperfusion injury in vitro Ischaemia- Understanding the kinetics of cell death following an insult can be very important to inform therapeutic interventions, as well as the underlying biology.

CellTox-Green enables kinetic measurement of cell death in cycles as short as 5min intervals (Fig. 1), or staggered over hours or days. This may be multiplexed with other kinetic dyes to maximise data output. For example - ROS, MPTP and Intracellular O2 dyes. Reading every 5 min for 24h causes little-to-no bleaching of the fluorophore. Cell tox green has been used at timepoints up to 72h in our lab. Fig 1: Rat myoblast cell death in response to 1h simulated ischaemiareperfusion injury. Kinetic measurement of chamber O2 (%) (Red) and cell death (blue) measured by CellTox-Green every 5min over a short ischaemiareperfusion protocol. Atmospheric gases were manipulated using Clariostar plate reader fitted with atmospheric control unit. Cell counting by automated immunofluorescence a) b)

Measuring cell death and DNA damage in response to olaparib treatment. Automated fluorescence microscopy with the Cytation (Biotek) of Hoechst 33342 staining allows for assaying nuclei following treatment. Multiples images per well of 96-well and 384-well plates can be stitched together to give whole-well data (Fig. 2a). Automated nuclei identification can be used as a measure of cell death (Fig. 2b). Multiplex with antibody staining (for example H2AX to H2AX to identify double stranded DNA breaks (Fig. 2c)) or endpoint mechanistic dyes (such as the MTMP dye Mitotraker Red) to investigate mechanisms of toxicity. c) Fig 2: Effect of Olaparib treatment in mesothelioma cells. Cancer cells were fixed with 4% PFA and stained with Hoechst 33324. 9

overlapping images per well were stitched together and deconvolved to form each image in (a). Automatic analysis counts cells and reports mean + standard deviation per sample (b). Representation of automated nuclei All assays can be performed in 96-well and 384-well format to increase throughput. All assays are suitable for both adherent and non-adherent cell culture. The DDG have several plate readers available for use. Easily perform synergy experiments between compound treatments (for example: test

compound + [Menadione]) Compound hit picking, serial dilution and cell treatments are automated to increase throughput and data reproducibility using the BRAVO (Agilent) liquid handling system. We are happy to discuss other assays not mentioned in this App note (for example the Annexin 5 Apoptosis/ Necrosis multiplex assay (Promega)). For questions and further information please contact: Richard Angell Drug discovery group lead; ([email protected]) Trevor Askwith Senior biologist; ([email protected]). Ben Allsop Biologist (Author); ([email protected]).

Recently Viewed Presentations

  • The Crucible - WordPress.com

    The Crucible - WordPress.com

    The Crucible, Scene One Understanding Questions Act One The Crucible, Scene One Analysis Questions Copy and complete the following table to show the various different attitudes towards witchcraft displayed in Scene 1. Some can appear contradictory; this is the wonder...
  • Overview of Enrollment for Education Solutions and Microsoft ...

    Overview of Enrollment for Education Solutions and Microsoft ...

    1ATA student use benefit is not available via CSP.M:EE student use benefit will not be available as a standalone in CSP, though it is included in the Microsoft 365 bundle student use benefits
  • Working Group 1 HECRTF Workshop June 14-16,2003

    Working Group 1 HECRTF Workshop June 14-16,2003

    Working Group 1 Enabling Technologies Chair: Sheila Vaidya Vice Chair: Stu Feldman WG 1 - Enabling Technologies Charter Charter Establish the basic technologies that may provide the foundation for important advances in HEC capability, and determine the critical tasks required...
  • Evolving CCMs to Deliver on the GF Strategy

    Evolving CCMs to Deliver on the GF Strategy

    CCMs in Challenging Contexts. Burundi, DRC, Haiti, Niger. The Evolution Pilot involved a . collective and consultative design and approach. Data analysis and consultations with over 260 partners, including 84 CCMs, Board and Committees, and internal GF Secretariat teams resulted...
  • View from the Top* - Transportation.org

    View from the Top* - Transportation.org

    Engaging in infrastructure-readiness promotional initiatives (such as the SPaT challenge), and. Supporting the installation of test beds that allow testing of AVs and CV applications in both controlled- as well as real world - environments. States are anticipating AVs and...
  • Starter of the day - Mrs. Francesconi's Art Class

    Starter of the day - Mrs. Francesconi's Art Class

    What is Art? Brainstorm on chalk board. Is graffiti art? Is a Lego brick art? Is trash art? Evidence that something is art. Composition is present in a piece. Art elements and principles of design are present in a piece....
  • Konkurranserett og statsstøtte

    Konkurranserett og statsstøtte

    Defrenne-sak EØF art 119 (EF art 141) Handleplikt - krever nasjonal gjennomføring. Likevel direkte virkning Private som pliktsubjekter Der traktaten er taus mht pliktsubjekt, sak 36/74 Walrave, sak C-281/98, Angonese Der staten angir staten som pliktsubjekt, sak 43/75, 2. Defrenne-sak...
  • Myths and Truths about the impact of palliative care programs ...

    Myths and Truths about the impact of palliative care programs ...

    Myths and Truths About Palliative Care. 1. Correct misunderstandings about the impact of palliative care programs on hospice programs. 2. Discuss evidence in the literature about how different kinds of palliative care programs influence hospice referrals, hospice length of stay,...